Fc-epsilon car

ABSTRACT

Recombinant NK cells, and especially recombinant NK-92 cells express a chimeric antigen receptor (CAR) having an intracellular domain of FcεRIγ. Notably, CAR constructs with an intracellular domain of FcεRIγ had a substantially prolonged duration of expression and significantly extended cytotoxicity over time. The CAR may be expressed from RNA and DNA, preferably as a tricistronic construct that further encodes CD16 and a cytokine to confer autocrine growth support. Advantageously, such constructs also enable high levels of transfection and expression of the recombinant proteins and provide a convenient selection marker to facilitate rapid production of recombinant NK/NK-92 cells.

This application claims the benefit of priority to allowed U.S. patentapplication with the Ser. No. 17/341,098, which was filed Jun. 7, 2021,which claims priority to U.S. patent application with the Ser. No.17/056,385, which was filed Nov. 17, 2020, which is a 371 application ofInternational application with the serial number PCT/US2019/033407,which was filed May 21, 2019, which claims priority to U.S. patentapplication with the Ser. No. 62/674,936, which was filed May 22, 2018,all of which are incorporated by reference herein.

SEQUENCE LISTING

The content of the XML file of the sequence listing named104077.0004US3.xml, which is 116 kb in size was created on Mar. 6, 2023and electronically submitted via Patent Center along with the presentapplication is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The field of the invention is recombinant nucleic acids and cellscontaining same to generate genetically modified cells that express achimeric antigen receptor (CAR), and particularly modified NK and NK-92cells expressing a CAR having an Fc epsilon receptor gamma (FcεRIγ)signaling domain.

BACKGROUND OF THE INVENTION

Natural killer (NK) cells are cytotoxic lymphocytes that constitute asignificant component of the innate immune system. In most cases, NKcells represent about 10-15% of circulating lymphocytes, and bind andkill targeted cells, including virus-infected cells and many malignantcells. NK cell killing is non-specific with regard to particularantigens and can occur without prior immune sensitization. Killing oftargeted cells is typically mediated by cytolytic proteins, includingperforin, granzyme, and granulysin.

Autologous NK cells have been used as therapeutic entities. To that end,NK cells are isolated from the peripheral blood lymphocyte fraction ofwhole blood, expanded in cell culture to obtain sufficient numbers ofcells, and then re-infused into a subject. Autologous NK cells haveshown in at least some cases moderate effectiveness in both ex vivotherapy and in vivo treatment. However, isolation and growth ofautologous NK cell is time and cost intensive. Moreover, autologous NKcell therapy is further limited by the fact that not all NK cells arecytolytic.

At least some of these difficulties can be overcome by use of NK-92cells, which are a cytolytic cancer cell line which was discovered inthe blood of a subject suffering from a non-Hodgkins lymphoma and thenimmortalized in vitro (Gong et al., Leukemia 8:652-658 (1994)). WhileNK-92 cells are NK cell derivatives, NK-92 cells lack the major ofinhibitory receptors that are otherwise displayed by normal NK cells,and retain the majority of the activating receptors. NK-92 cells do not,however, attack normal cells nor do they elicit an unacceptable immunerejection response in humans. Due to these desirable characteristics,NK-92 cells were characterized in detail and explored as therapeuticagent in the treatment of certain cancers as is described, for example,in WO 1998/049268 or US 2002/068044.

Phenotypic changes distinguishing a tumor cell from normal cells derivedfrom the same tissue are often associated with one or more changes inthe expression of specific gene products, including the loss of normalcell surface components or the gain of others (i.e., antigens notdetectable in corresponding normal, non-cancerous tissue). The antigenswhich are expressed in neoplastic or tumor cells, but not in normalcells, or which are expressed in neoplastic cells at levelssubstantially above those found in normal cells, have been termed“tumor-specific antigens” or “tumor-associated antigens.” Suchtumor-specific antigens may serve as markers for tumor phenotype.Tumor-specific antigens include cancer/testis-specific antigen (e.g.MAGE, BAGE, GAGE, PRAME and NY-ESO-1), melanocyte differentiationantigens (e.g. tyrosinase, Melan-A/MART, gp100, TRP-1 and TRP-2),mutated or aberrantly expressed antigens (e.g. MUM-1, CDK4,beta-catenin, gp100-in4, p15 and N-acetylglucos-aminyltransferase V),and antigens that are expressed at higher levels in tumors (e.g., CD19and CD20).

Tumor-specific antigens have been used as targets for cancerimmunotherapies. One such therapy utilizes chimeric antigen receptors(CARs) expressed on the surface of immune cells, including T cells andNK cells, to improve cytotoxicity against cancer cells. CARs comprise asingle-chain variable fragment (scFv) linked to at least oneintracellular signaling domain. The scFv recognizes and binds an antigenon the target cell (e.g., a cancer cell) and triggers effector cellactivation. The signaling domains contain immunoreceptor tyrosine-basedactivation domains (ITAMs) that are important for intracellularsignaling by the receptor.

The first generation of CARs used in T-cells contained one cytoplasmicsignaling domain. For example, one version of a first-generation CAR inT-cells included a signaling domain from the Fc epsilon receptor gamma(FcεRIγ) which contained one ITAM, while another version contained thesignaling domain from CD3ζ which contained three ITAMs. In vivo and invitro studies showed that the CD3ζ CAR T-cells were more efficient attumor eradication than FcεRIγ CAR T-cells (e.g., Haynes, et al. 2001, J.Immunology 166:182-187; Cartellieri, et al. 2010, J. Biomed and Biotech,Vol. 2010, Article ID 956304). Additional studies then revealed thatcertain costimulatory signals were required for full activation andproliferation of such recombinant T-cells, and second and thirdgeneration CARs combined multiple signaling domains in to a single CARto enhance efficacy of the recombinant CAR T-cells. Due to their lessdesirable philological effects in the tested T-cells, first generationCARs and the FcεRIγ signaling domains were largely discarded in favor ofthe new, more efficient CARs using CD3ζ in combination with one or moreadditional signaling domains (e.g., Hermanson and Kaufman 2015,Frontiers in Immunol., Vol. 6, Article 195).

More recently, selected CARs have also been expressed in NK cells. Forexample, CAR-modified NK-92 cells have used first generation CARs withonly a CD3ζ intracellular signaling domain. Several antigens have beentargeted by these first generation CAR-NK cells, including CD19 and CD20for B cell lymphoma, ErbB2 for breast, ovarian, and squamous cellcarcinoma, GD2 for neuroblastoma, and CD138 for multiple myeloma. Secondgeneration CAR-NK cells from the NK-92 line have also been created forseveral antigens, including EpCAM for multiple carcinomas HLA-A2 EBNA3complex for Epstein-Barr virus, CS1 for multiple myeloma, and ErbB2 forHER2 positive epithelial cancers. The most common intracellularcostimulatory domain used alongside CD3ζ in second generation NK-92 CARsis CD28. However, the potential effect of the CD28 domain is unclearsince NK cells do not naturally express CD28. Additional secondgeneration CARs have incorporated the 4-1BB intracellular signalingdomain along with CD3ζ to improve NK cell persistence. Others comparedfunctionality of different intracellular domains using an ErbB2 scFvfused with CD3ζ alone, CD28 and CD3ζ, or 4-1BB and CD3ζ tested againstbreast cancer cells. They found that both of the second generationconstructs improved killing compared to the first generation CARs andthe CD28 and CD3ζ had 65% target lysis, the 4-1BB and CD3ζ lysed 62%,and CD3ζ alone killed 51% of targets. 4-1BB and CD28 intracellulardomains were also compared in a recent study using anti-CD19 CARsexpressed on NK-92 cells for B cell malignances. Still others found thatCD3ζ/4-1BB constructs were less effective than CD3ζ/CD28 in cell killingand cytokine production, highlighting differential effects of CD28 and4-1BB costimulatory domains.

Third generation NK-92 CARs were constructed of an anti-CD5 scFv withCD3ζ, CD28, and 4-1BB intracellular signaling domains and demonstratedspecific and potent anti-tumor activity against a variety of T-cellleukemia and lymphoma cell lines and primary tumor cells. Such cellswere also able to inhibit disease progression in xenograft mouse modelsof T cell Acute lymphoblastic leukemia (ALL) cell lines as well asprimary tumor cells (Transl Res. 2017 September; 187: 32-43). In furtherexamples, WO 2016/201304 and WO 2018/076391 teach use of thirdgeneration CD3ζ CARs expressed in NK cells and NK-92 cells.

Autologous NK cells and NK-92 cells require exogenous IL-2 as a survivalfactor and enhancer of cytotoxic potential. Unfortunately, systemicadministration of IL-2 is often associated with significant undesirableside effects and toxicity. To overcome such issues, the cells can becultivated and expanded in vitro prior to administration to a patient.While IL-2 will allow generation of sufficient quantities of NK cells orNK-92 cells, use of exogenous IL-2 in large scale production of NK cellsis typically cost-prohibitive. The requirement for exogenous IL-2 wasresolved by recombinant expression of IL-2 confined to the endoplasmicreticulum from a retroviral vector (see Exp Hematol. 2005 February;33(2):159-64). Such approach eliminated the requirement for exogenousIL-2. However, retroviral transfection efficiency is often less thandesirable and will be even more inefficient where multiple recombinantgenes are to be expressed.

In addition, NK cells and particularly NK-92 cells are often difficultto genetically modify as evidenced by numerous failures to engineerNK-92 cells to express an Fc receptor. Such difficulties are furthercompounded where NK-92 cells are transfected with multiple recombinantgenes or relatively large recombinant nucleic acid payload forheterologous expression. Additionally, NK-92 cells also exhibit asignificant lack of predictability with respect to recombinantexpression of exogenous proteins (e.g., CD16). On a functional level,most if not all CAR NK-92 cells require a relatively high effector totarget cell ratio, likely due to relatively low expression of the CARconstruct. Moreover, such CAR NK-92 cells will also experience a fastdecline in cytotoxicity over time, thus rendering such cells clinicallyless attractive.

Therefore, even though numerous recombinant NK-92 cells are known in theart, all or almost all of them suffer from various difficulties.Consequently, there remains a need for CAR-expressing NK-92 cells thatexpress a high-activity CAR in significant quantities with attendantpersistent cytotoxicity, and that are easily cultivated in a simple andeffective manner.

SUMMARY OF THE INVENTION

The inventors have discovered that NK-92 cells can be efficientlytransfected with a recombinant nucleic acid to express anFcεRIγ-containing CAR. Unexpectedly, CARs with a FcεRIγ signaling domainsignificantly increased expression levels of the CAR and furtherconveyed extended cytotoxicity over time. Contemplated recombinantnucleic acids that encode a CAR are preferably in a tricistronicarrangement that also includes a sequence portion that encodes CD16 orCD16 variant, and/or IL-2 or an IL-2 variant. Advantageously, suchrecombinant nucleic acids not only provide an efficient manner ofselecting transfected cells (as the IL-2 not only imparts autocrinegrowth stimulation but also acts as a selection marker for theco-expressed proteins), but also yield CAR NK cells with superiorcytolytic activity (e.g., at a relatively low effector to target cellratio as compared to other constructs) and high levels of expression ofthe CD16 and the FcεRIγ-containing CAR.

Therefore, in one aspect of the inventive subject matter, the inventorscontemplate a genetically modified NK cell that recombinantly expressesa cytokine, CD16, and a membrane bound chimeric antigen receptor (CAR).The CAR will typically comprise in a single polypeptide chain (i) anextracellular binding domain, (ii) a hinge domain, (iii) a transmembranedomain, and (iv) a FcεRIγ signaling domain (e.g., having the amino acidsequence of SEQ ID NO:1).

In many embodiments, the NK cell is an NK-92 cell, and/or therecombinantly expressed cytokine is or comprises IL-2 or IL-15 (whichmay further include an endoplasmic retention sequence). In furtherembodiments, the CD16 may be a high-affinity CD16 variant (e.g.,CD16_(158V)).

Preferably, but not necessarily, the extracellular binding domain willcomprise a scFv that may specifically bind to a tumor-specific antigen(e.g., CD19, CD20, NKG2D ligands, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C,GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met,EGFR, FAP, WT-1, PSMA, NY-ESO1, CSPG-4, IGF1-R, Flt-3, CD276, CD123,PD-L1, BCMA, or CD33), a tumor associated antigen, or a patient- andtumor-specific antigen, or that may specifically bind to avirus-specific antigen (e.g., antigen of an HIV virus, an HPV virus, anRSV virus, an influenza virus, an ebolavirus, or an HCV virus).

In some embodiments, the cytokine, the CD16, and the CAR are expressedfrom a tricistronic recombinant nucleic acid, while in other embodimentsthe cytokine and/or the CD16 is expressed from a recombinant nucleicacid that is integrated into the genome of the NK cell.

Therefore, the inventors also contemplate a recombinant nucleic acidthat includes a first sequence portion encoding a cytokine, a secondsequence portion encoding a CD16, and a third sequence portion encodinga chimeric antigen receptor (CAR) that comprises in a single polypeptidechain an extracellular binding domain, a hinge domain, a transmembranedomain, and an FcεRIγ signaling domain. Most typically, the first, thesecond, and the third sequence portions are on the same nucleic acid.

While in some embodiments the nucleic acid is a tricistronic RNA, inother embodiments the nucleic acid is a tricistronic DNA.

Moreover, it is typically preferred that the cytokine is IL-2 or IL15(which may or may not comprise an endoplasmic retention sequence), thatthe CD16 is a high-affinity CD16 variant having a 158V mutation, and/orthat the extracellular binding domain comprises a scFv. As noted before,the extracellular binding domain may specifically bind to atumor-specific antigen, a tumor associated antigen, or a patient- andtumor-specific antigen, or the extracellular binding domain mayspecifically bind to a virus-specific antigen.

In further contemplated aspects, the hinge domain and/or thetransmembrane domain comprise a CD8 hinge domain and/or a CD28transmembrane domain, while the FcεRIγ signaling domain may have anucleic acid sequence of SEQ ID NO:2.

In still further aspects of the inventive subject matter, the inventorsalso contemplate a recombinant cell comprising the recombinant nucleicacid described above and herein. Where the nucleic acid is preparedand/or amplified, the recombinant cell may be a bacterial cell. On theother hand, where the recombinant nucleic acid is to be expressed, thecell will typically be an autologous NK cell or an NK cell (which mayalso be an NK-92 cell that is optionally genetically modified).

Consequently, the inventors also contemplate a method of treating cancerin a patient in need thereof. In such method, a therapeuticallyeffective amount of any one of the genetically modified NK cells isadministered to the patient, thereby treating the cancer. In addition,and where desired, contemplated methods may include a further step ofadministering at least one additional therapeutic entity selected fromthe group consisting of a viral cancer vaccine, a bacterial cancervaccine, a yeast cancer vaccine, N-803, an antibody, a stem celltransplant, and a tumor targeted cytokine.

Among other cancers, contemplated cancers include leukemia, acutelymphocytic leukemia, acute myelocytic leukemia, chronic leukemias,chronic myelocytic (granulocytic) leukemia, chronic lymphocyticleukemia, polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin'sdisease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chaindisease, solid tumors including, but not limited to, sarcomas andcarcinomas such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovariancancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilm's tumor, cervical cancer, testicular tumor, lungcarcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma.

Likewise, the inventors also contemplate a method of treating a viralinfection in a patient in need thereof. In such method, atherapeutically effective amount of any one of the genetically modifiedNK cells is administered to the patient, thereby treating the viralinfection. Where desired or needed, an antiviral drug may also beadministered.

Regardless of the type of treatment, it is generally contemplated that1×10⁸ to about 1×10¹¹ cells per m2 of body surface area of the patientare administered to the patient. Viewed from a different perspective,use of a genetically modified NK cell as presented herein iscontemplated in the treatment of cancer or a viral infection.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments along with the accompanying drawingfigures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of exemplary CD19-CARs tested. Allof the CD19-CAR variants contained an extracellular domain comprising ananti-CD19 scFv region (αCD19-scFv), a hinge region from CD8 (CD8 hinge),and a transmembrane domain from CD28 (CD28 TM). The intracellulardomains of the CD19CARs were varied as indicated.

FIG. 2A are exemplary results for the percentage of NK-92 cellsexpressing the CD19-CAR of FIG. 1 after transfection with CD19-CAR mRNAas determined by flow cytometry with an anti-scFv antibody labeled witheF660.

FIG. 2B are exemplary results for the median fluorescent intensity (MFI)minus background for CD19-CAR-expressing NK-92 cells labeled with ananti-scFv antibody labeled with eF660.

FIG. 3A shows exemplary results for the percentage of NK-92cell-sensitive target cancer cells (K562) that were killed by NK-92cells (effector) expressing the CD19CARs at effector:target ratios offrom 5:1 to 0.3:1.

FIG. 3B shows exemplary results for the percentage of NK-92cell-resistant, CD19-positive target cancer cells (SUP-B15) that werekilled by NK-92 cells (effector) expressing the CD19CARs ateffector:target ratios of from 5:1 to 0.3:1.

FIG. 4 shows exemplary results for the MFI of CD19-CAR-expressing NK-92cells (effector) labeled with anti-CD107a antibody in a degranulationassay with SUP-B15 target cells at effector:target ratios of from 2:1 to0.25:1.

FIG. 5 shows exemplary results for surface expression of CD19 CAR onhaNK cells transfected with CD19 CAR mRNA constructs at various timepoints. All CAR constructs tested show detectable expression for up to72 h under the conditions used with CD19/CD28-Fc-epsilon CAR having thelongest duration of expression.

FIG. 6 shows exemplary results for cytotoxicity of CD19.taNK on SUPB15CD19+ cells (an aNK resistant cell line). All CAR constructs tested showcomparable (maximum) cytotoxic properties at 24 h. However, at 48 h,CD19/CD3-zeta shows a marked decrease in cytotoxic properties whileFc-epsilon based CARs show only minimal decrease 48 hourspost-electroporation.

FIG. 7 is an exemplary schematic of a recombinant tricistronic DNAconstruct and corresponding protein products.

FIG. 8 shows an exemplary linearized version of the plasmid shown inFIG. 8 .

FIG. 9A shows exemplary results for in vitro data showing that CD33positive (CD33+) THP-1 cells are relatively resistant to cytotoxicity(specific lysis) by control NK-92 (aNK) cells, whereas there is a highpercentage of specific lysis when THP-1 cells are cultured with NK-92cells that express a CAR that specifically binds CD33 (CD33-CAR/NK-92cells).

FIG. 9B shows exemplary results for in vitro data showing that K562cells are killed by both control aNK cells and CD33-CAR/NK-92 cells.

FIG. 10 shows exemplary results for cytotoxicity of HER2.CAR-t-haNKcells against BT-474 cells.

FIG. 11 shows exemplary results for cytotoxicity of CD33.CAR-t-haNKcells against THP-1 cells.

FIG. 12 shows exemplary results for cytotoxicity of PD-L1.CAR-t-haNKcells against SUP-B15.PD-L1′⁺ cells.

FIG. 13 shows exemplary results for cytotoxicity of PD-L1.CAR-t-haNKcells against U251 cells.

FIG. 14 shows exemplary results for cytotoxicity of EGFR.CAR-t-haNKcells against A-549 cells.

FIG. 15 shows exemplary results for cytotoxicity of CD19.CAR-t-haNKcells against K562 cells.

FIG. 16 shows exemplary results for cytotoxicity of CD19.CAR-t-haNKcells against SUP-B15 cells.

FIG. 17 shows exemplary results for ADCC of CD19.CAR-t-haNK cellsagainst SKBr3 cells.

FIG. 18 shows exemplary results for cytotoxicity of IGF1R.CAR-t-haNKcells against MDA-MB-231 cells.

FIG. 19 shows exemplary results for cytotoxicity of PD-L1.CAR-t-haNKcells against a variety of cancer cells.

FIG. 20 shows exemplary comparative results for cytotoxicity ofPD-L1.CAR-t-haNK cells against MDA-MB-231 cells.

FIG. 21 shows exemplary results expression of CD16 and CD19.CAR.

FIG. 22 shows exemplary results for natural cytotoxicity ofCD19.CAR-t-haNK cells against K562 cells.

FIG. 23 shows exemplary results for CAR mediated cytotoxicity ofCD19.CAR-t-haNK cells against SUP-B15 cells.

FIG. 24 shows exemplary results for ADCC of CD19.CAR-t-haNK cells.

FIG. 25 shows exemplary comparative results for expression of CD16 andCD20.CAR.

FIG. 26 shows exemplary results for natural cytotoxicity ofCD20.CAR-t-haNK cells.

FIG. 27 shows exemplary results for expression of CD16 and CD33.CAR.

FIG. 28 shows exemplary results for natural cytotoxicity ofCD33.CAR-t-haNK cells against K562 cells.

FIG. 29 shows exemplary results for CAR mediated cytotoxicity ofCD33.CAR-t-haNK cells against THP-1 cells.

FIG. 30 shows exemplary results for ADCC of CD33.CAR-t-haNK cells.

FIG. 31 shows exemplary results for expression of CD16 and EGFR.CAR.

FIG. 32 shows exemplary results for natural cytotoxicity ofEGFR.CAR-t-haNK cells against K562 cells.

FIG. 33 shows exemplary results for CAR mediated cytotoxicity ofEGFR.CAR-t-haNK cells against A549 cells.

FIG. 34 shows exemplary results for CAR mediated cytotoxicity ofEGFR.CAR-t-haNK cells against HCT116 cells.

FIG. 35 shows exemplary results for ADCC of EGFR.CAR-t-haNK cells.

FIG. 36 shows exemplary results for expression of CD16 and HER2.CAR.

FIG. 37 shows exemplary results for natural cytotoxicity ofHER2.CAR-t-haNK cells against K562 cells.

FIG. 38 shows exemplary results for CAR mediated cytotoxicity ofHER2.CAR-t-haNK cells against SKBR-3 cells.

FIG. 39 shows exemplary results for ADCC of HER2.CAR-t-haNK cells.

FIG. 40 shows exemplary results expression of CD16 and PD-L1.CAR.

FIG. 41 shows exemplary results for natural cytotoxicity ofPD-L1.CAR-t-haNK cells against K562 cells.

FIG. 42 shows exemplary results for CAR mediated cytotoxicity ofPD-L1.CAR-t-haNK cells.

FIG. 43 shows exemplary results for ADCC of PD-L1.CAR-t-haNK cells.

FIG. 44 shows exemplary results for CAR mediated cytotoxicity ofCD123.CAR-t-haNK cells.

FIG. 45 shows exemplary results for ADCC of CD123.CAR-t-haNK cells.

FIG. 46 shows exemplary results for expression of CD16 and CD30.CAR.

FIG. 47 shows exemplary results for natural cytotoxicity ofCD30.CAR-t-haNK cells against K562 cells.

FIG. 48 shows exemplary results for CAR mediated cytotoxicity ofCD30.CAR-t-haNK cells against THP-1 cells.

FIG. 49 shows exemplary results for ADCC of CD30.CAR-t-haNK cells.

FIG. 50 shows exemplary results for CD16 and BCMA.CAR expression.

FIG. 51 shows exemplary results for CAR mediated cytotoxicity ofBCMA.CAR-t-haNK cells.

FIG. 52 shows exemplary results for ADCC of BCMA.CAR-t-haNK cells.

FIG. 53 shows exemplary results for expression of CD16 and gp120.CAR.

FIG. 54 shows exemplary results for GP120 binding of gp120.CAR-t-haNKcells.

FIG. 55 shows exemplary results for natural cytotoxicity ofgp120.CAR-t-haNK cells against K562 cells.

FIG. 56 shows exemplary results for ADCC of gp120.CAR-t-haNK cells.

FIG. 57 shows exemplary results for CD16 and FAP.CAR expression.

FIG. 58 shows exemplary results for CAR mediated cytotoxicity ofFAP.CAR-t-haNK cells.

FIG. 59 shows exemplary results for CSPG4 expression in CSPG4.CAR-t-haNKcells.

FIG. 60 shows exemplary results for CAR mediated cytotoxicity ofCSPG4.CAR-t-haNK cells against SK-MEL-28 cells.

FIG. 61 depicts an exemplary tricistronic construct encoding IGF1R-CAR,CD16, and IL-2^(ER).

DETAILED DESCRIPTION OF THE INVENTION

The inventors have unexpectedly discovered that CAR mediatedcytotoxicity and CAR expression in recombinant NK cells (e.g. NK-92cells) is substantially increased where the recombinant CAR includes anFcεRIγ signaling domain as is described in more detail below. Thefinding that a CAR with an FcεRIγ signaling domain has superiorproperties in NK cells is especially unexpected as such CARs in T cellshave performed relatively poorly as compared to CARs that had a CD3ζ, a4-1BB, or a CD28 signaling domain and optionally additional signalingdomains as commonly found in second and third generation CARs.

Therefore, in some embodiments recombinant nucleic acids arecontemplated that encode a CAR with an FcεRIγ signaling domain,preferably but not necessarily in a tricistronic arrangement that alsoincludes a sequence portion that encodes CD16 or a CD16 variant, and/orIL-2 or an IL-2 variant. In still further advantageous aspects of theinventive subject matter, such recombinant nucleic acid will not onlyprovide an efficient manner of selecting transfected cells (as the IL-2not only imparts autocrine growth stimulation) but also acts as aselection marker for the co-expressed proteins.

Consequently, the inventive subject matter is directed to geneticallymodified NK cells, NK-92 cells, and derivatives thereof that express achimeric antigen receptor (CAR) on the cell surface where the CARpreferably comprises an intracellular signaling domain from the Fcepsilon receptor gamma (FcεRIγ). For example, the cytoplasmic domain ofFcεRIγ may have an amino acid sequence having at least 95% sequenceidentity to SEQ ID NO:1, or comprises, consists of, or essentiallyconsists of an amino acid sequence having the sequence as noted in SEQID NO:1. In some embodiments, the cytoplasmic domain of FcεRIγ isencoded by a nucleic acid having at least 95% sequence identity to SEQID NO:2. Contemplated recombinant cells may further express variousother proteins, including one or more cytokines and CD16. As will bereadily appreciated, the CAR and/or other proteins may be transientlyexpressed by the recombinant cell, or stably expressed.

In some embodiments, the CAR comprises a hinge region from CD8 and/or insome embodiments, the CAR comprises a transmembrane domain from CD28having an amino acid sequence as in SEQ ID NO:6 (encoded by a nucleicacid as in SEQ ID NO:7). The full length amino acid sequence of CD28 isshown in SEQ ID NO:23. In further embodiments, the recombinant cell isgenetically modified with a nucleic acid having a sequence of SEQ IDNO:9 that encodes a hybrid protein having a sequence of SEQ ID NO:8comprising a CD8 hinge region that is coupled to a CD28 transmembranedomain that is coupled to an FcεRIγ signaling domain. As will beappreciated, addition of a binding domain to the hinge region will forma functional CAR. For example, binding domain targets or specificallymay bind a tumor-associated antigen, and suitable antigens include CD19,CD20, NKG2D ligands, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244,CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAP,WT-1, PSMA, NY-ESO1, CSPG-4, IGF1-R, Flt-3, CD276, CD123, PD-L1, BCMA,and CD33.

In some embodiments, the nucleic acid construct further comprises a(inducible) promoter that promotes transcription of the nucleic acidsequences. Preferably, but not necessarily, the nucleic acid constructis a multi-cistronic vector or RNA comprising one or more InternalRibosome Entry Site (IRES) to allow for initiation of translation froman internal region of an mRNA transcribed from the nucleic acidsequences. Alternatively, or additionally, the nucleic acid constructcomprises a sequence that encodes a 2A peptide, such as a T2A, P2A, E2A,or F2A peptide, in order to produce equimolar levels of polypeptidesencoded by the same mRNA. In some embodiments, the nucleic acidconstruct further comprises a nucleic acid sequence that encodes anantigen binding protein (ABP). In some embodiments, the ABP is an scFvor a codon optimized scFv. In some embodiments, the ABP specificallybinds an antigen expressed by a tumor cell. In some embodiments, the ABPis part of a chimeric antigen receptor (CAR). In further embodiments,the construct comprises a nucleic acid that encodes a cytokine, suchIL-2 or IL-15, which may be targeted to the endoplasmic reticulum. Insome embodiments, the NK-92 cell or cell line is also geneticallymodified to express CD16 on the cell surface. In one embodiment, theNK-92 cell or cell line is genetically modified to express a highaffinity CD16 (F158V) on the cell surface.

With respect to suitable NK cells, it should be noted that all NK cellsare deemed suitable for use herein and therefore include primary NKcells (preserved, expanded, and/or fresh cells), secondary NK cells thathave been immortalized, autologous or heterologous NK cells (banked,preserved, fresh, etc.), and modified NK cells as described in moredetail below. In some embodiments, it is preferred that the NK cells areNK-92 cells. The NK-92 cell line is a unique cell line that wasdiscovered to proliferate in the presence of interleukin 2 (IL-2) (seee.g., Gong et al., Leukemia 8:652-658 (1994)). NK-92 cells are cancerousNK cells with broad anti-tumor cytotoxicity and predictable yield afterexpansion in suitable culture media. Advantageously, NK-92 cells havehigh cytolytic activity against a variety of cancers.

The original NK-92 cell line expressed the CD56bright, CD2, CD7, CD11a,CD28, CD45, and CD54 surface markers and did not display the CD1, CD3,CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD20, CD23, and CD34 markers.Growth of such NK-92 cells in culture is dependent upon the presence ofinterleukin 2 (e.g., rIL-2), with a dose as low as 1 IU/mL beingsufficient to maintain proliferation. IL-7 and IL-12 do not supportlong-term growth, nor have various other cytokines tested, includingIL-la, IL-6, tumor necrosis factor α, interferon α, and interferon γ.Compared to primary NK cells, NK-92 typically have a high cytotoxicityeven at relatively low effector:target (E:T) ratios, e.g. 1:1.Representative NK-92 cells are deposited with the American Type CultureCollection (ATCC), designation CRL-2407.

Therefore, suitable NK cells may have one or more modified KIR that aremutated such as to reduce or abolish interaction with MHC class Imolecules. Of course, it should be noted that one or more KIRs may alsobe deleted or expression may be suppressed (e.g., via miRNA, siRNA,etc.). Most typically, more than one KIR will be mutated, deleted, orsilenced, and especially contemplated KIR include those with two orthree domains, with short or long cytoplasmic tail. Viewed from adifferent perspective, modified, silenced, or deleted KIRs will includeKIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1,KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, andKIR3DS1. Such modified cells may be prepared using protocols well knownin the art. Alternatively, such cells may also be commercially obtainedfrom NantKwest (see URL www.nantkwest.com) as aNK cells (‘activatednatural killer cells). Such cells may then be additionally geneticallymodified to a CAR as further described in more detail below.

In another aspect of the inventive subject matter, the geneticallyengineered NK cell may also be an NK-92 derivative that is modified toexpress the high-affinity Fcγ receptor (CD16). Sequences forhigh-affinity variants of the Fcγ receptor are well known in the art(see e.g., Blood 2009 113:3716-3725), and all manners of generating andexpression are deemed suitable for use herein. Expression of suchreceptor is believed to allow specific targeting of tumor cells usingantibodies that are specific to a patient's tumor cells (e.g.,neoepitopes), a particular tumor type (e.g., her2neu, PSA, PSMA, etc.),or that are associated with cancer (e.g., CEA-CAM). Advantageously, suchantibodies are commercially available and can be used in conjunctionwith the cells (e.g., bound to the Fcγ receptor). Alternatively, suchcells may also be commercially obtained from NantKwest as haNK cells.Such cells may then be additionally genetically modified to a CAR asfurther described in more detail below.

Genetic modification of the NK cells contemplated herein can beperformed in numerous manners, and all known manners are deemed suitablefor use hereon. Moreover, it should be recognized that NK cells can betransfected with DNA or RNA, and the particular choice of transfectionwill at least in part depend on the type of desired recombinant cell andtransfection efficiency. For example, where it is desired that NK cellsare stably transfected, linearized DNA may be introduced into the cellsfor integration into the genome. On the other hand, where transienttransfection is desired, circular DNA or linear RNA (e.g., mRNA withpolyA+ tail) may be used.

For example, where the NK cell is an autologous NK cell or an NK-92 cellit is contemplated that the recombinant nucleic acid will include asegment that encodes a CAR that includes FcεRIγ signaling domain, andpreferably also a segment that encodes a cytokine to provide autocrinegrowth stimulation (e.g., IL-2, IL-2 that is modified with an ERretention sequence, IL-15, or IL-15 that is modified with an ERretention sequence) and/or a segment that encodes a CD16 or highaffinity CD16¹⁵⁸v. As will be readily appreciated, inclusion of acytokine that provides autocrine growth stimulation will render themodified recombinant independent of exogenous cytokine addition, whichwill render large scale production of such cells economically feasible.Likewise, where the modified recombinant also expresses CD16 or a highaffinity CD16^(158V), such cells will have further enhanced ADCCcharacteristics and with that further improved targeted cytotoxicity.

Of course, it should be recognized that the recombinant nucleic acidthat encodes that cytokine and/or the CD16 or high affinity CD16^(158V)can be integrated in to the genome of the NK cell, or can be supplied asan extrachromosomal unit (which may be a linear or circular DNA, or alinear RNA, virally delivered or via chemical, mechanical, or electricaltransfection). For example, recombinant NK-92 cells expressing IL-2ERand CD16158V are known as haNK cells (Oncotarget 2016 Dec. 27; 7(52):86359-86373) and can be transfected with a recombinant nucleic acid thatincludes a segment that encodes a CAR that includes FcεRIγ signalingdomain. Once more, such recombinant nucleic acid may comprise furthersegments that may encode additional immunotherapeutic proteins, such asN-803, TxM-type compounds, IL-8 traps, TGF-β traps, etc. Likewise, NK-92cells may already be transfected with a cDNA that encodes IL-2 (e.g.,NK-92MI, ATCC CRL-2408). Such cells can then be further transfected witha recombinant nucleic acid that includes a segment that encodes a CARthat includes FcεRIγ signaling domain along with a segment that encodesa CD16 or high affinity CD16^(158V).

On the other hand, (autologous, fresh, cultivated, or previously frozen)NK cells or NK-92 cells may also be transfected with a recombinantnucleic acid that includes a segment that encodes a CAR with a FcεRIγsignaling domain, a segment that encodes a cytokine to provide autocrinegrowth stimulation (e.g., IL-2, IL-2 that is modified with an ERretention sequence, IL-15, or IL-15 that is modified with an ERretention sequence) and a segment that encodes a CD16 (SEQ ID NO:34) orhigh affinity CD16^(158V) (SEQ ID NO:35, encoded by SEQ ID NO:36). Mosttypically, such recombinant nucleic acid will be arranged as atricistronic construct. As noted before, such constructed can be anextrachromosomal circular plasmid, a linear DNA (which may be integratedinto the genome of the NK cell), or a linear RNA. Such nucleic acidswill typically be transfected into the cells in a manner well known inthe art (e.g., electroporation, lipofection, ballistic gene transfer,etc.). Similarly, the nucleic acid may be delivered to the cell via arecombinant virus. Therefore, NK cells suitable for use herein includeNK-92 cells (which may be transfected with a tricistronic constructencoding a CAR, a CD16 or variant thereof, and a cytokine or variantthereof), a genetically modified NK cell or NK-92 cell that expresses aCD16 or variant thereof or a cytokine or variant thereof (which may betransfected with a nucleic acid encoding a CAR and a CD16 or variantthereof or a cytokine or variant thereof), and a genetically modified NKcell or NK-92 cell that expresses a CD16 or variant thereof and acytokine or variant thereof (which may be transfected with a nucleicacid encoding a CAR).

In preferred embodiments, it should therefore be noted that thegenetically modified NK cell (especially where the cell expresses a CARand CD16 or variant thereof) will exhibit three distinct modes of cellkilling: General cytotoxicity which is mediated by activating receptors(e.g., an NKG2D receptor), ADCC which is mediated by antibodies bound toa target cell, and CAR mediated cytotoxicity.

Consequently, it should be appreciated that the manner of transfectionwill at least in part depend on the type of nucleic acid employed.Therefore, viral transfection, chemical transfection, mechanicaltransfection methods are all deemed suitable for use herein. Forexample, in one embodiment, the vectors described herein are transientexpression vectors. Exogenous transgenes introduced using such vectorsare not integrated in the nuclear genome of the cell; therefore, in theabsence of vector replication, the foreign transgenes will be degradedor diluted over time.

In another embodiment, the vectors described herein allow for stabletransfection of cells. In one embodiment, the vector allowsincorporation of the transgene(s) into the genome of the cell.Preferably, such vectors have a positive selection marker and suitablepositive selection markers include any genes that allow the cell to growunder conditions that would kill a cell not expressing the gene.Non-limiting examples include antibiotic resistance, e.g. geneticin (Neogene from Tn5).

Alternatively, or additionally, the vector is a plasmid vector. In oneembodiment, the vector is a viral vector. As would be understood by oneof skill in the art, any suitable vector can be used, and suitablevectors are well-known in the art.

In still other embodiments, the cells are transfected with mRNA encodingthe protein of interest (e.g., the CAR). Transfection of mRNA results intransient expression of the protein. In one embodiment, transfection ofmRNA into NK-92 cells is performed immediately prior to administrationof the cells. In one embodiment, “immediately prior” to administrationof the cells refers to between about 15 minutes and about 48 hours priorto administration. Preferably, mRNA transfection is performed about 5hours to about 24 hours prior to administration. In at least someembodiments as described in more detail below, NK cell transfection withmRNA resulted in unexpectedly consistent and strong expression of theCAR at a high faction of transfected cells. Moreover, such transfectedcells also exhibited a high specific cytotoxicity at comparably loweffector to target cell ratios.

With respect to contemplated CARs it is noted that the NK or NK-92 cellswill be genetically modified to express the CAR as a membrane boundprotein exposing a portion of the CAR on the cell surface whilemaintaining the signaling domain in the intracellular space. Mosttypically, the CAR will include at least the following elements (inorder): an extracellular binding domain, a hinge domain, a transmembranedomain, and an FcεRIγ signaling domain.

In preferred embodiments, the cytoplasmic domain of the CAR comprises orconsists of a signaling domain of FcεRIγ. Notably, and as described inmore detail below, the FcεRIγ signaling domain provide for substantiallyincreased expression levels of the CAR as much as for significantlyextended cytotoxicity over time. For example, the FcεRIγ signalingdomain comprises or consists of or consists essentially of the aminoacid sequence of SEQ ID NO:1. In some embodiments, the FcεRIγcytoplasmic domain is the sole signaling domain. However, it should beappreciated that additional elements may also be included, such as othersignaling domains (e.g., CD28 signaling domain, CD3ζ signaling domain,4-1BB signaling domain, etc.). These additional signaling domains may bepositioned downstream of the FcεRIγ cytoplasmic domain and/or upstreamof the FcεRIγ cytoplasmic domain.

In some embodiments, the FcεRIγ signaling domain comprises or consistsof or consists essentially of an amino acid sequence having at leastabout 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology to theamino acid sequence of SEQ ID NO:1.

In alternative embodiments, the cytoplasmic domain of the CAR may alsocomprise a signaling domain of CD3 zeta (CD3ζ). In one embodiment, thecytoplasmic domain of the CAR consists of a signaling domain of CD3zeta. In one embodiment, the CD3 zeta signaling domain comprises orconsists of or consists essentially of the amino acid sequence of SEQ IDNO:15. In some embodiments, the CD3 zeta signaling domain comprises orconsists of or consists essentially of an amino acid sequence having atleast about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology tothe amino acid sequence of SEQ ID NO:15.

The CAR may comprise any suitable transmembrane domain. In one aspect,the CAR comprises a transmembrane domain of CD28. In one embodiment, theCD28 transmembrane domain comprises or consists of or consistsessentially of the amino acid sequence of SEQ ID NO:6 (encoded bynucleic acid with the SEQ ID NO:7). In one embodiment, the CD28transmembrane domain comprises or consists of or consists essentially ofan amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%,98%, or 99% sequence homology to the amino acid sequence of SEQ ID NO:6.In other embodiments, the transmembrane domain may also be a 4-1BBtransmembrane domain.

The CAR may comprise any suitable hinge region. In one aspect, the CARcomprises a hinge region of CD8. In one embodiment, the CD8 hinge regioncomprises or consists of or consists essentially of the amino acidsequence of SEQ ID NO:3 or SEQ ID NO:4. In one embodiment, the CD8 hingeregion comprises or consists of or consists essentially of an amino acidsequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence homology to the amino acid sequence of SEQ ID NO:3 or SEQ IDNO:4. Such region may be encoded by a nucleic acid having the sequenceof SEQ ID NO: 5.

Therefore, contemplated CARs will include a general structure of adesired antigen binding domain that is coupled to a hinge domain, whichis coupled to a transmembrane domain, which is coupled to a signalingdomain. Viewed from another perspective, contemplated CARs may have adesired binding domain that is then coupled to a hybrid protein thatcomprises, consists of, or essentially consists of a hinge domain, whichis coupled to a transmembrane domain, which is coupled to a signalingdomain. For example, such hybrid protein may have an amino acid sequencehaving at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequencehomology to the amino acid sequence of SEQ ID NO:8 (encoded by nucleicacid sequence SEQ ID NO:9).

Most typically, but not necessarily, the extracellular binding domain ofthe CAR will be a scFv or other natural or synthetic binding portionthat specifically binds an antigen of interest. Especially suitablebinding portions include small antibody fragments with single, dual, ormultiple target specificities, beta barrel domain binders, phage displayfusion proteins, etc. Among other suitable extracellular bindingdomains, preferred domains will specifically bind to a tumor-specificantigen, a tumor associated antigen, or a patient- and tumor-specificantigen. Tumor-specific antigens include, without limitation, NKG2Dligands, CS1, GD2, CD 138, EpCAM, EBNA3 C, GPA7, CD244, CA-125, ETA,MAGE, CAGE, BAGE, HAGE, LAGE, PAGE, NY-SEO-1, GAGE, CEA, CD52, CD30,MUC5AC, c-Met, EGFR, FAP, WT-l, PSMA, NY-ESO1, AFP, CEA, CTAG1B, andCD33. Additional non-limiting tumor-associated antigens, and themalignancies associated therewith, can be found in Table 1. Stillfurther tumor-specific antigens are described, by way of non-limitingexample, in US2013/0189268; WO 1999024566 A1; U.S. Pat. No. 7,098,008;and WO 2000020460, each of which is incorporated herein by reference inits entirety. Likewise, other preferred domains will specifically bindto a (pathogenic) virus-specific antigen, such as an antigen of an HIVvirus (e.g., gp120), an HPV virus, an RSV virus, an influenza virus, anebolavirus, or an HCV virus.

TABLE 1 Target antigen Associated malignancy α-Folate receptor Ovariancancer CAIX Renal cell carcinoma CD19 B-cell malignancies CLL B-ALL ALL;ALL post-HSCT Lymphoma; Refractory Follicular Lymphoma; B-NHL LeukemiaB-cell malignancies; B-cell malignancies post-HSCT B-lineage lymphoidmalignancies post-UCBT B-cell malignancies, CLL, B-NHL CD19/CD20Lymphoblastic leukemia CD20 Lymphomas B-cell malignancies B-celllymphomas Mantle cell lymphoma indolent B-NHL Leukemia CD22 B-cellmalignancies CD30 Lymphomas; Hodgkin lymphoma CD33 AML CD44v7/8 Cervicalcarcinoma CD138 Multiple myeloma CD244 Neuroblastoma CEA Breast cancerColorectal cancer CS1 Multiple myeloma EBNA3C EBV positive T cells EGP-2Multiple malignancies EGP-40 Colorectal cancer EpCAM Breast carcinomaerb-B2 Colorectal cancer Breast and others Prostate cancer erb-B 2, 3, 4Breast and others FBP Ovarian cancer Fetal acetylcholine receptorRhabdomyosarcoma GD2 Neuroblastoma GD3 Melanoma GPA7 Melanoma Her2Breast carcinoma Ovarian cancer Tumors of epithelial origin Her2/neuMedulloblastoma Lung malignancy Advanced osteosarcoma GlioblastomaIL-13R-a2 Glioma Glioblastoma Medulloblastoma KDR Tumor neovasculaturek-light chain B-cell malignancies (B-NHL, CLL) LeY Carcinomas Epithelialderived tumors L1 cell adhesion molecule Neuroblastoma MAGE-A1 MelanomaMesothelin Various tumors MUC1 Breast, Ovary NKG2D ligands Varioustumors Oncofetal antigen (h5T4) Various tumors PSCA Prostate carcinomaPSMA Prostate/tumor vasculature TAA targeted by mAb IgE Various tumorsTAG-72 Adenocarcinomas VEGF-R2 Tumor neovasculature

For example, the CAR may comprise an anti-CD19 extracellular domain. Inone embodiment, the anti-CD19 extracellular domain comprises, consistsof, or consists essentially of the amino acid sequence of SEQ ID NO:11.In one embodiment, the anti-CD19 extracellular domain comprises orconsists of or consists essentially of an amino acid sequence having atleast about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology tothe amino acid sequence of SEQ ID NO:11.

Consequently, contemplated CARs will target antigens associated with aspecific cancer type. For example, targeted cancers include leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, solid tumors including, but notlimited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovariancancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilm's tumor, cervical cancer, testicular tumor, lungcarcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Therefore, contemplated CARs will generally have a structure of anextracellular binding domain that is (directly) coupled to a hingedomain, which is (directly) coupled to a transmembrane domain, which is(directly) coupled to an FcεRIγ signaling domain. In still furthercontemplated aspects, contemplated CARs may also include one or moresignaling domains in addition to or replacing the FcεRIγ signalingdomain, and especially contemplated signaling domains include CD3ζsignaling domains, 4-1BB signaling domains, and CD28 signaling domains.For example, contemplated CARs may therefore include any binding domain(e.g., having SEQ ID NO:11) that is coupled to a hinge domain (e.g., CD8hinge as in SEQ ID NO:3 or SEQ ID NO:4, encoded by SEQ ID NO:5), whichis in turn coupled to a transmembrane domain (e.g., CD28 TM as in SEQ IDNO:6, encoded by SEQ ID NO:7), which is coupled to a signaling domain(e.g., FcεRIγ signaling domain as in SEQ ID NO:1, encoded by SEQ IDNO:1, or CD28 signaling domain as in SEQ ID NO:13, or 4-1BB signalingdomain as in SEQ ID NO:14, or CD3ζ signaling domain as in SEQ ID NO:15)

With respect to the construction of contemplated CARs it should berecognized that CARs can be engineered in numerous manners as described,for example, in WO 2014/039523; US 2014/0242701; US 2014/0274909; US2013/0280285 and WO 2014/099671, each of which is incorporated herein byreference in its entirety.

In still further contemplated aspects, and as noted above, NK cells maybe further genetically modified to express one or more cytokines to soprovide a selection marker where the cytokine and the CAR are encoded onthe same recombinant nucleic acid, and/or to render the recombinantcells independent of exogenous IL-2. Therefore, in some aspects of theinventive subject matter, NK-92 cells are modified to express at leastone cytokine. In particular, the at least one cytokine is IL-2, IL-12,IL-15, IL-18, IL-21, or a variant thereof. In preferred embodiments, thecytokine is IL-2 or a variant thereof and especially preferred variantsinclude endoplasmic retention signals (e.g., human IL-2 as in SEQ IDNO:21, or with ER retention signal as in SEQ ID NO:22, SEQ ID NO:30, orSEQ ID NO:33). For example, the IL-2 gene is cloned and expressed with asignal sequence that directs the IL-2 to the endoplasmic reticulum. Thispermits expression of IL-2 at levels sufficient for autocrineactivation, but without releasing IL-2 extracellularly (e.g., ExpHematol. 2005 February; 33(2):159-64.) Alternatively, expression of acytokine (and especially IL-15) may also be such that the cytokine willbe expressed in sufficient quantities to provide an autocrine growthsignal to the recombinant cells, but also to allow at least some of theexpressed IL-15 to be released from the cell, which will so provide animmune stimulatory signal. For example, such expression may be achievedusing a human IL-15 sequence that includes both the signal peptide andan endoplasmic retention sequence. An exemplary DNA and protein sequencefor an endoplasmic retained IL-15 is shown in SEQ ID NO:49 and SEQ IDNO:50, respectively.

Where desired, contemplated cells may also express a suicide gene. Theterm “suicide gene” refers to a transgene that allows for the negativeselection of cells expressing the suicide gene. A suicide gene is usedas a safety system, allowing cells expressing the gene to be killed byintroduction of a selective agent. This is desirable in case therecombinant gene causes a mutation leading to uncontrolled cell growth,or the cells themselves are capable of such growth. A number of suicidegene systems have been identified, including the herpes simplex virusthymidine kinase (TK) gene, the cytosine deaminase gene, thevaricella-zoster virus thymidine kinase gene, the nitroreductase gene,the Escherichia coli gpt gene, and the E. coli Deo gene. Typically, thesuicide gene encodes for a protein that has no ill effect on the cellbut, in the presence of a specific compound, will kill the cell. Thus,the suicide gene is typically part of a system.

In one embodiment, the suicide gene is active in NK-92 cells. In oneembodiment, the suicide gene is the thymidine kinase (TK) gene. The TKgene may be a wild-type or mutant TK gene (e.g., tk30, tk75, sr39tk).Cells expressing the TK protein can be killed using ganciclovir. Inanother embodiment, the suicide gene is cytosine deaminase, which istoxic to cells in the presence of 5-fluorocytosine. Garcia-Sanchez etal. “Cytosine deaminase adenoviral vector and 5-fluorocytosineselectively reduce breast cancer cells 1 million-fold when theycontaminate hematopoietic cells: a potential purging method forautologous transplantation.” Blood. 1998 Jul. 15; 92(2):672-82. In afurther embodiment, the suicide gene is cytochrome P450, which is toxicin the presence of ifosfamide or cyclophosphamide. See, e.g. Touati etal. “A suicide gene therapy combining the improvement ofcyclophosphamide tumor cytotoxicity and the development of an anti-tumorimmune response.” Curr Gene Ther. 2014; 14(3):236-46. In yet anotherembodiment, the suicide gene is iCasp9. Di Stasi, (2011) “Inducibleapoptosis as a safety switch for adoptive cell therapy.” N Engl J Med365: 1673-1683. See also Morgan, “Live and Let Die: A New Suicide GeneTherapy Moves to the Clinic” Molecular Therapy (2012); 20: 11-13. iCasp9induces apoptosis in the presence of a small molecule, AP1903. AP1903 isbiologically inert small molecule, that has been shown in clinicalstudies to be well tolerated, and has been used in the context ofadoptive cell therapy.

Of course, it should be noted that all of the recombinant proteins canbe expressed from individual recombinant sequences. However, it isgenerally preferred that where multiple recombinant sequences areexpressed (e.g., CAR, CD16, cytokine), coding regions may be arranged ina polycistronic unit with at least two or at least three coding regionsencoding the recombinant proteins. For example, a tricistronic DNA orRNA construct (e.g., encoding a CAR with an FcεRIγ signaling domain, aCD16^(158V), and IL-2^(ER) or IL15^(ER)) may be transfected into an NKor NK-92 cell. Therefore, transgenes can be engineered into anexpression vector by any mechanism known to those of skill in the art.Where multiple transgenes are to be inserted into a cell, transgenes maybe engineered into the same expression vector or a different expressionvector. In some embodiments, the cells are transfected with mRNAencoding the transgenic protein to be expressed. In some embodiments,the cells are transfected with DNA encoding the transgenic protein to beexpressed. Transgenes, mRNA and DNA can be introduced into the NK-92cells using any transfection method known in the art, including, by wayof non-limiting example, infection, viral vectors, electroporation,lipofection, nucleofection, or “gene-gun.”

As will be readily apparent, contemplated genetically modified cells canbe used for treatment of various diseases, and especially of variouscancers and viral infections where a diseased cell presents adisease-specific or disease-associated antigen. Consequently, theinventors contemplate methods of treating patients with modified NK orNK-92 cells as described herein. In one embodiment, the patient issuffering from cancer (e.g., a tumor) and the modified NK-92 cell orcell line expresses a CAR specific for an antigen expressed on thesurface of a cell from the cancer or tumor. In one embodiment, thepatient is suffering from a viral infection and the modified NK-92 cellor cell line expresses a CAR specific for an antigen expressed on thesurface of a cell that has been infected by the virus. In oneembodiment, the patient is suffering from a bacterial infection and themodified NK-92 cell or cell line expresses a CAR specific for an antigenexpressed on the surface of a bacterial cell causing the infection.

Contemplated modified NK or NK-92 cells can be administered to anindividual by absolute numbers of cells. For example, the individual canbe administered from about 1000 cells/injection to up to about 10billion cells/injection, such as at about, at least about, or at mostabout, 1×10⁸, 1×10⁷, 5×10⁷, 1×10⁶, 5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴,1×10³, 5×10³ (and so forth) modified NK-92 cells per injection, or anyranges between any two of the numbers, end points inclusive. In otherembodiments, modified NK-92 cells can be administered to an individualby relative numbers of cells, e.g., said individual can be administeredabout 1000 cells to up to about 10 billion cells per kilogram of theindividual, such as at about, at least about, or at most about, 1×10⁸,1×10⁷, 5×10⁷, 1×10⁶, 5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴, 1×10³, 5×10³(and so forth) modified NK-92 cells per kilogram of the individual, orany ranges between any two of the numbers, end points inclusive. Inother embodiments, the total dose may calculated by m² of body surfacearea, including about 1×10¹¹, 1×10¹⁰, 1×10⁹, 1×10⁸, 1×10⁷, per m², orany ranges between any two of the numbers, end points inclusive. Theaverage person is about 1.6 to about 1.8 m². In a preferred embodiment,between about 1 billion and about 3 billion NK-92 cells are administeredto a patient.

The modified NK-92 cells, and optionally other anti-cancer or anti-viralagents can be administered once to a patient with cancer or infectedwith a virus or can be administered multiple times, e.g., once every 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22 or 23 hours, or once every 1, 2, 3, 4, 5, 6 or 7 days, or once every1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks during therapy, or anyranges between any two of the numbers, end points inclusive.

In one embodiment, where the modified NK-92 cells express a suicidegene, the patient is administered an agent to trigger modified NK-92cell death. In one embodiment, the agent is administered at a time pointafter administration of the modified NK-92 cells that is sufficient forthe NK-92 cells to kill target cells.

In one embodiment, the modified NK-92 cells are irradiated prior toadministration to the patient. Irradiation of NK-92 cells is described,for example, in U.S. Pat. No. 8,034,332, which is incorporated herein byreference in its entirety. In one embodiment, modified NK-92 cells thathave not been engineered to express a suicide gene are irradiated.

Furthermore, it should be appreciated that contemplated treatments willalso include administration of other immune therapeutic entities, andespecially preferred immune therapeutic entities include a viral cancervaccine (e.g., adenoviral vector encoding cancer specific antigens), abacterial cancer vaccine (e.g., non-pyrogenic E. coli expressing one ormore cancer specific antigens), a yeast cancer vaccine, N-803 (alsoknown as ALT-803, ALTOR Biosciences), an antibody (e.g., binding to atumor associated antigen or patient specific tumor neoantigen), a stemcell transplant (e.g., allogeneic or autologous), and a tumor targetedcytokine (e.g., NHS-IL12, IL-12 coupled to a tumor targeting antibody orfragment thereof).

EXAMPLES

The following examples are for illustrative purposes only and should notbe interpreted as limitations of the claimed invention. There are avariety of alternative techniques and procedures available to those ofskill in the art which would similarly permit one to successfullyperform the intended invention.

Example 1: CAR mRNA Preparation

DNA sequences encoding each variant of CD19CAR schematically depicted inFIG. 1 were designed in silico, synthesized de novo, and subcloned intothe mRNA expression vector, pXT7 (GeneArt, Life Technologies). Tenmicrograms (μg) of plasmid were linearized by digestion with the SalIrestriction enzyme (New England Biolabs) and purified using a QIAgen gelpurification kit (QIAgen) according to manufacturer's instructions.

The linearized DNA was used as template for in vitro synthesis of mRNAusing a T7 mMessage mMachine Ultra transcription kit (ThermoFisherScientific, Waltham, Mass.) according to the manufacturer'sinstructions. This kit includes a polyadenylation extension step thatincreases the length of the polyA tail of the mRNA and thus enhancesstability in vivo.

mRNA for six CD19CAR variants were prepared, with a green fluorescentprotein (GFP) mRNA prepared as a negative control. All of the CD19CARvariants contained an extracellular domain comprising an anti-CD19 scFvregion (αCD19-scFv) (SEQ ID NO:11), a hinge region from CD8 (SEQ ID NO:3or NO:4), and a transmembrane domain from CD28 (SEQ ID NO:6 encoded bySEQ ID NO:7) The intracellular domains of the CD19CARs were as followsand schematically shown in FIG. 1 : CAR 3z contained a CD3ζ signalingdomain; CAR FcRe contained a FcεRIγ signaling domain (SEQ ID NO:1); CAR28_3z contained a CD28 signaling domain fused to a CD3ζ signalingdomain; CAR BB_3z contained a 4-1BB signaling domain fused to a CD3ζsignaling domain; CAR 28_BB_3z contained a CD28 signaling domain fusedto a 4-1BB signaling domain fused to a CD3ζ signaling domain; CARBB_3z_28 contained a 4-1BB signaling domain fused to a CD3ζ signalingdomain fused to a CD28 signaling domain.

More particularly, the 1^(st) generation CAR with CD3ζ signaling domainof FIG. 1 had a nucleic acid sequence of SEQ ID NO:16 (human). The1^(st) generation CAR with a FcεRIγ signaling domain nucleic had anucleic acid sequence of SEQ ID NO:12 and an amino acid sequence of SEQID NO:10. The 2^(nd) generation CAR with CD28/CD3ζ signaling domain hada nucleic acid sequence of SEQ ID NO:17 and the 2^(nd) generation CARwith 4-1BB/CD3ζ signaling domain had a nucleic acid sequence of SEQ IDNO:18. The 3^(rd) generation CAR with CD28/4-1BB/CD3ζ signaling domainhad a nucleic acid sequence of SEQ ID NO:19 and the 3^(rd) generationCAR with 4-1BB/CD3ζ/CD28signaling domain had a nucleic acid sequence ofSEQ ID NO:20. A further 1^(st) generation CAR with a FcεRIγ signalingdomain nucleic had an amino acid sequence of SEQ ID NO:25.

Example 2: Electroporation of NK-92 Cells with CD19CAR mRNA

NK-92 cells were grown in X-Vivol0 medium (Lonza, Basel, Switzerland)supplemented with 5% Human AB Serum (Valley Biomedical, Winchester, Va.)and 500 IU/mL IL-2 (Prospec, Rehovot, Israel). Cells were electroporatedwith mRNA using the Neon™ electroporation device (Life Technologies,Carlsbad, Calif.), following the manufacturer's parameters for NK-92cells (1250 V, 10 ms, 3 pulses) and using 5 μg of mRNA per 10⁶ cells ina volume of 100 μl. Electroporated cells were maintained in medium (sameas above) for 20 hours (h).

The CD19CAR expression on the NK-92 cell surface was determined by flowcytometry using anti-scFv antibody labeled with eF660 (eBioscience, SanDiego, Calif.). FIG. 2A shows the % expression of the indicated CD19CARin the NK-92 cell population. FIG. 2B shows the median fluorescenceintensity (MFI, minus background) of cells electroporated with theindicated CD19CAR. As can be taken from FIGS. 2A and 2B, CAR FcReunexpectedly had the highest percentage of cells (75.2%) expressingCD19CAR at the cell surface, as well as the highest MFI (quantity ofexpressed CAR on a recombinant cell), followed by 28_3z (61.7%).

Example 3: Cytotoxicity of NK-92 Cells Expressing CD19CAR Against CancerCell Lines

The efficacy of CAR-expressing NK-92 cells to target cancer cells invitro was tested 20 hours post-electroporation using a flow-based invitro cytotoxicity assay. Effector cells (NK-92 expressing CD19CAR orGFP) were mixed with PKHGL67-labeled (Sigma-Aldrich, St. Louis, Mo.)target cells (K562; or SUPB15, B-ALL, CD19⁺) at different effector totarget ratios (5:1 to 0.3:1) in a 96-well plate and incubated 4 h at 37°C. Propidium Iodide (PI) (Sigma Aldrich, St. Louis, Mo.) was added tothe cells and samples were analyzed within 2 h using an Attune flowcytometer (Life Technologies, Carlsbad, Calif.). The cytotoxicity wasdetermined by the % of PI-positive cells within the PKH-positive targetpopulation.

Exemplary results are provided in FIGS. 3A and 3B. NK-92 cells areeffective at killing K562 cells regardless of CD19CAR expression as canbe seen from FIG. 3A. Thus, it should be noted that recombinant cellswill not lose cytotoxicity. In contrast, GFP-expressing NK-92 cells wereinefficient at killing the cancer cell line SUP-B15. SUP-B15 is an acutelymphoblastic leukemia cell line that is CD19-positive and resistant toNK-92-mediated cytotoxicity. Expression of any CD19CAR tested providedincreased cytotoxic activity against the SUP-B15 cell line compared tocontrol (GFP-expressing NK-92 cells) as can be readily taken from FIG.3B. Surprisingly, CARs with the FcεRIγ signaling domain exhibitedcytotoxicity similar or even superior to the 2^(nd) and 3^(rd)generation CARs. Such finding is particularly unexpected as the FcεRIγsignaling domain was present only as a single unit and not combined withother signaling domains. Such arrangement, when used in CAR T-cellsfailed to provide desirable targeted cytotoxicity.

Degranulation is a critical step required for the release of the lyticproteins (e.g., perforin and granzyme) from secretory granules in theNK-92 cells. Degranulation is initiated by recognition of a target cellby NK-92. To test degranulation in the constructs, effector cells(NK-92) were mixed with unlabeled target cells (SUP-B15) at differenteffector to target ratios (5:1 to 0.3:1) in a 96-well plate, andanti-CD107a (FITC-conjugated, BD Pharmingen, San Jose, Calif.) was addedto each well. Plates were incubated at 37° C. in a CO₂ incubator andafter 1 h monensin (Golgi-stop) was added to the wells. The plates wereincubated for another 3 h at 37° C. and the samples were analyzed byflow cytometry (Attune, Life technologies, Carlsbad, Calif.). Percentagedegranulation was determined by subtracting the % CD107a positive inNK-92 cells alone to the % CD107a positive in the effector+targetsamples, and exemplary results are provided in FIG. 4 .

Example 4. Surface Expression and Cytotoxicity of NK-92 Cells ExpressingCD19CAR Against Cancer Cell Lines

The inventors quantified expression levels for the various CARconstructs to investigate durability of expression over time. As can beseen from the results in FIG. 5 , NK-92 cells transfected with thedifferent CD19 CAR constructs expressed detectable levels of therespective CARs on the cell surface for up to 72 hours. Unexpectedly,and as can be readily seen from FIG. 5 , the CAR constructs thatcomprised the Fc-epsilon cytoplasmic signaling domain had substantiallyhigher durations of expression. Notably, it was also observed thataddition of one or more signaling domains in addition to the FcεRIγsignaling domain (e.g., CD28 signaling domain in the example presentedhere) would not adversely affect the duration of expression. Indeed, inthe CAR having the FcεRIγ signaling domain and the CD28 signaling domainduration of expression was even further increased over time, whereas CARconstructs with a CD3-zeta signaling domain had a dramatic reduction inexpression at the 72 hour mark, and even before then.

Moreover, as can also be seen from the results in FIG. 5 , the quantityof expression of CAR constructs having the FcεRIγ signaling domain wasalso initially significantly higher than corresponding constructs with aCD3-zeta signaling domain.

The inventors then set out to test whether the extended and strongerexpression of the CAR constructs having the FcεRIγ signaling domainwould also translate into a higher rate of cytotoxicity. Exemplaryresults for tests on SUPB15 CD19⁺ cells at 24 hours and 48 hours aredepicted in FIG. 6 . As can be taken from the results, all CARconstructs tested showed somewhat comparable (maximum) cytotoxicproperties at 24 hours. However, at 48 hours, CD19/CD3-zeta showed amarked decrease in cytotoxic properties. Remarkably, the Fc-epsilonbased CARs showed only minimal decrease in cytotoxic activity 48 hourspost-electroporation, which paralleled the extended expression resultsfrom FIG. 5 . Thus, it should be recognized that the CAR constructs withan FcεRIγ signaling domain exhibited extended cytotoxicity, which isbelieved to be of substantial clinical benefit.

Advantageously, tricistronic mRNA constructs were able to producesubstantial quantities of desired CARs with excellent functionalactivity. Such constructs are especially beneficial where the CARexpression should be transient. In contrast, the following examples fortargeted CAR constructs and associated functional data were fromlinearized DNA vector constructs, which allowed transfected cells tointegrate the linearized DNA into the genome and to so provide an avenuefor non-transient expression of the specific CARs.

Example 5. Map of Tricistronic Expression Cassette

FIG. 7 shows diagrammatically the DNA and protein products produced by arepresentative tricistronic expression cassette. FIG. 8 shows thelinearized version of the plasmid with the expression cassette.

SEQ ID NO:28 is an exemplary nucleic acid sequence for part of thepNEUKv1_CD19CAR_CD16(158V)_ERIL-2 vector, which is a construct similarto FIG. 8 . SEQ ID NO:29 is an exemplary tricistronic protein (similarto FIG. 7 ) that represents a CD19CAR_P2A_CD16(158V) protein. SimilarlySEQ ID NO:31 is an exemplary nucleic acid sequence for theCodon-optimized CD33ScfV-P2A-CD16-IRES-erIL2 tricistronic sequence,while SEQ ID NO:32 shows a CD33 CAR-P2A-CD16 peptide.

Still further constructs made include SEQ ID NO:24 is an exemplary aminoacid sequence for CD19K_Transmembrane and Signaling domain, while SEQ IDNO.:26 is an exemplary nucleic acid sequence for15AD23HC_1805843_CD19K_Eps (879-1319), and SEQ ID NO: 27 is an exemplarynucleic acid sequence for 15AD23HC_1805843_CD19K_Eps, which did notinclude a CD28 transmembrane domain.

Example 6. Cytotoxicity of NK-92 Cells Expressing CD33-CAR AgainstCancer Cell Lines

The following example is provided to demonstrate that cells that areresistant to specific lysis (cytotoxicity) by control (unmodified) NK-92cells can be efficiently killed by NK-92 cells that express a CAR. Inthis example, the cells were THP-1 cells expressing CD33. NK-92 cellwere modified to express a CAR with an extracellular binding domainspecifically binds to CD33, and that an FcεRIγ signaling domain as shownin FIGS. 8 and 9 .

FIG. 9A provides in vitro data showing that CD33 positive (CD33+) THP-1cells are relatively resistant to cytotoxicity (specific lysis) bycontrol NK-92 (aNK) cells, whereas there is a high percentage ofspecific lysis when THP-1 cells are cultured with NK-92 cells thatexpress a CAR that specifically binds CD33 (CD33-CAR/NK-92 cells).Moreover, it should be noted that the modified NK-92 cells expressingthe CAR exhibited killing at a relatively low effector:target ratio.FIG. 9B provides in vitro data showing that K562 cells are efficientlykilled by both control aNK cells and CD33-CAR/NK-92 cells.

Example 7: HER2-CAR with FcεRIγ Signaling Domain

In this example, the inventors constructed a 1^(st) generation CARs witha FcεRIγ signaling domain that included an anti-HER2 scFv coupled to aCD8 hinge, that in turn was coupled to a CD28 transmembrane domain,which was coupled to a FcεRIγ signaling domain. The so constructedHER2-CAR had a nucleic acid sequence of SEQ ID NO:37.

Functionality of the so constructed HER2.CAR-t-haNK cells was testedagainst BT-474 cells using a standard CalceinAM-based cytotoxicity assayand exemplary results are shown in FIG. 10 . As can be readily seen fromthe data, the HER2.CAR-t-haNK cells expressing the CAR with the FcεRIγsignaling domain exhibited significant cytotoxicity against the BT-474target cells.

In further experiments, the inventor demonstrated expression of theHER2.CAR in HER2.CAR-t-haNK cells as is illustrated in FIG. 36 . Naturalcytotoxicity of the HER2.CAR-t-haNK cells is shown in the results ofFIG. 37 , while results for CAR mediated cytotoxicity are shown in FIG.38 . Exemplary data for ADCC of HER2.CAR-t-haNK cells are shown in thegraph of FIG. 39 .

Example 8: CD30-CAR with FcεRIγ Signaling Domain

In this example, the inventors constructed a 1^(st) generation CARs witha FcεRIγ signaling domain that included an anti-CD30 scFv coupled to aCD8 hinge, that in turn was coupled to a CD28 transmembrane domain,which was coupled to a FcεRIγ signaling domain. The so constructedCD30-CAR had a nucleic acid sequence of SEQ ID NO:38.

Expression of the CD30-CAR is demonstrated in the results of FIG. 46 ,while the results for natural cytotoxicity of the recombinant cells areshown in FIG. 47 . CAR mediated cytotoxicity was demonstrated in theresults of FIG. 48 , while exemplary results for ADCC are shown in thedata of FIG. 49 .

Example 9: EGFR-CAR with FcεRIγ Signaling Domain

In this example, the inventors constructed a 1^(st) generation CARs witha FcεRIγ signaling domain that included an anti-EGFR scFv coupled to aCD8 hinge, that in turn was coupled to a CD28 transmembrane domain,which was coupled to a FcεRIγ signaling domain. The so constructedEGFR-CAR had a nucleic acid sequence of SEQ ID NO:39.

Functionality of the so constructed EGFR.CAR-t-haNK cells was testedagainst A-549 cells using a standard cytotoxicity assay and exemplaryresults are shown in FIG. 14 . As can be readily seen from the data, theEGFR.CAR-t-haNK cells expressing the CAR with the FcεRIγ signalingdomain exhibited significant cytotoxicity against the A-549 targetcells. Expression of the EGFR-CAR in the EGFR.CAR-t-haNK cells is shownin FIG. 31 , while natural cytotoxicity results are shown in FIG. 32 .Exemplary results for CAR mediated cytotoxicity of EGFR.CAR-t-haNK cellsare shown in FIG. 33 and FIG. 34 , while results for ADCC ofEGFR.CAR-t-haNK cells are shown in FIG. 35 .

Example 10: IGF1R-CAR with FcεRIγ Signaling Domain

In this example, the inventors constructed a 1^(st) generation CARs witha FcεRIγ signaling domain that included an anti-IGF1R scFv coupled to aCD8 hinge, that in turn was coupled to a CD28 transmembrane domain,which was coupled to a FcεRIγ signaling domain. The so constructedIGF1R-CAR had a nucleic acid sequence of SEQ ID NO:40, and atricistronic construct encoding IGF1R-CAR, CD16, and IL-2ER had anucleic acid sequence of SEQ ID NO:53, which is also schematicallyillustrated in FIG. 61 .

Functionality of the so constructed IGF1R.CAR-t-haNK cells was testedagainst MDA-MB-231 cells using a standard cytotoxicity assay incomparison with a 2^(nd) generation CAR (CD28/CD3z) and exemplaryresults are shown in FIG. 18 . As can be readily seen from the data, theIGF1R.CAR-t-haNK cells expressing the CAR with the FcεRIγ signalingdomain exhibited significant and target specific cytotoxicity againstthe MDA-MB-231 target cells, which was comparable with the cytotoxicityof the 2^(nd) generation CAR.

Example 11: CD123-CAR with FcεRIγ Signaling Domain

In this example, the inventors constructed a 1^(st) generation CARs witha FcεRIγ signaling domain that included an anti-CD123 scFv coupled to aCD8 hinge, that in turn was coupled to a CD28 transmembrane domain,which was coupled to a FcεRIγ signaling domain. The so constructedCD123-CAR had a nucleic acid sequence of SEQ ID NO:41. Data for the CARmediated cytotoxicity of the CD123-CAR expressing recombinant NK cellsis shown in FIG. 44 , and FIG. 45 shows exemplary data for ADCC ofCD123-CAR expressing recombinant NK cells.

Example 12: PD-L1-CAR with FcεRIγ Signaling Domain

In this example, the inventors constructed a 1^(st) generation CARs witha FcεRIγ signaling domain that included an anti-PD-L1 scFv coupled to aCD8 hinge, that in turn was coupled to a CD28 transmembrane domain,which was coupled to a FcεRIγ signaling domain. The so constructedPD-L1-CAR had a nucleic acid sequence of SEQ ID NO:42.

Functionality of the so constructed PD-L1.CAR-t-haNK cells was testedagainst SUP-B15.PD-L1⁺ cells using a standard cytotoxicity assay andexemplary results are shown in FIG. 12 . As can be readily seen from thedata, the PD-L1.CAR-t-haNK cells expressing the CAR with the FcεRIγsignaling domain exhibited significant cytotoxicity against theSUP-B15.PD-L1⁺ target cells.

Functionality of the so constructed PD-L1.CAR-t-haNK cells was alsotested against U251 cells using a standard cytotoxicity assay andexemplary results are shown in FIG. 13 along with non-transfected haNKcells. As can be readily seen from the data, the PD-L1.CAR-t-haNK cellsexpressing the CAR with the FcεRIγ signaling domain exhibited targetspecific and significant cytotoxicity against the U251 target cells,whereas the haNK control cells had substantially no cytotoxicity againstthe same U251 cells.

In still further experiments on target cell specificity with respect toPD-L1, the inventors tested several PD-L1 positive tumor cell linesusing the PD-L1.CAR-t-haNK cells along with haNK cells as control forgeneral cytotoxicity. As can be readily seen from FIG. 19 , thePD-L1.CAR-t-haNK cells had superior cytotoxicity across a wide varietyof tumor cells (lung, breast, genitourinary tumor cells, andadditionally, head and neck small cell cancer, chordoma). Notably, thePD-L1.CAR-t-haNK cells required less than 4 hours for the majority(>85%) of cell killing whereas the control haNK cells required more than12 hours.

FIG. 20 further illustrates cytotoxicity of the PD-L1.CAR-t-haNK cellsagainst MDA-MB-231 cells as compared to various other control cells(haNK cells as indicated). As can be taken from the data, at a 5:1 E:Tratio, MDA-MB-231 lysis by PD-L1.thaNK was improved by cetuximab, andhaNK activity was improved by the addition of cetuximab and a-PD-L1.Plain PD-L1.thank had improved cytotoxic activity compared to haNK andhaNK+cetuximab, and plain PD-L1.thank killing was comparable to that ofhaNK+PD-L1 antibody but PD-L1.thank+cetuximab outperformedhaNK+cetuximab and haNK+PD-L1. At a 1:1 E:T ratio, PD-L1.thaNK activitywas the same with or without cetuximab, and PD-L1.thaNK significantlyoutperformed intrinsic and ADCC-mediated killing by hank. haNK activitywas improved by the addition of cetuximab and a-PD-L1.

In further experiments, the inventors demonstrated expression of thePD-L1.CAR in PD-L1.CAR-t-haNK cells as is illustrated in FIG. 40 .Natural cytotoxicity of the PD-L1.CAR-t-haNK cells is shown in theresults of FIG. 41 , while results for CAR mediated cytotoxicity areshown in FIG. 42 . Exemplary data for ADCC of PD-L1.CAR-t-haNK cells areshown in the graph of FIG. 43 .

Example 13: CD33-CAR with FcεRIγ Signaling Domain

In this example, the inventors constructed a 1^(st) generation CARs witha FcεRIγ signaling domain that included an anti-HER2 scFv coupled to aCD8 hinge, that in turn was coupled to a CD28 transmembrane domain,which was coupled to a FcεRIγ signaling domain. The so constructedCD33-CAR had a nucleic acid sequence of SEQ ID NO:43.

Functionality of the so constructed CD33.CAR-t-haNK cells was testedagainst THP-1 cells using a standard cytotoxicity assay and exemplaryresults are shown in FIG. 11 . As can be readily seen from the data, theCD33.CAR-t-haNK cells expressing the CAR with the FcεRIγ signalingdomain exhibited significant cytotoxicity against the THP-1 targetcells. Further data depicting strong expression of the CD33CAR in NK-92cells are presented in FIG. 27 . Natural cytotoxicity of theCD33.CAR-t-haNK cells against K562 cells is shown in FIG. 28 , and FIG.29 depicts results for CAR mediated cytotoxicity against THP-1 cells.FIG. 30 shows further results for ADCC of CD33.CAR-t-haNK cells againstSUP-B15 CD19^(KO)/CD20⁺ with rituximab.

Example 14: gp120-CAR with FcεRIγ Signaling Domain

In this example, the inventors constructed a 1^(st) generation CARs witha FcεRIγ signaling domain that included an anti-gp120 scFv coupled to aCD8 hinge, that in turn was coupled to a CD28 transmembrane domain,which was coupled to a FcεRIγ signaling domain. The so constructedgp120-CAR had a nucleic acid sequence of SEQ ID NO:44.

The inventors further demonstrated that so generated cells expressedsignificant quantities of CD16 and gp120CAR as can be seen from FIG. 53. Binding of GP120 to the gp120CAR was shown as demonstrated in FIG. 54versus non-recombinant aNK cells as negative control. Naturalcytotoxicity of the so generated cells is shown in FIG. 55 , whilecorresponding ADCC data are shown in FIG. 56 .

Example 15: B7-H4-CAR with FcεRIγ Signaling Domain

In this example, the inventors constructed a 1^(st) generation CARs witha FcεRIγ signaling domain that included an anti-B7-H4 scFv coupled to aCD8 hinge, that in turn was coupled to a CD28 transmembrane domain,which was coupled to a FcεRIγ signaling domain. The so constructedB7-H4-CAR had a nucleic acid sequence of SEQ ID NO:45.

Example 16: BCMA-CAR with FcεRIγ Signaling Domain

In this example, the inventors constructed a 1^(st) generation CARs witha FcεRIγ signaling domain that included an anti-BCMA scFv coupled to aCD8 hinge, that in turn was coupled to a CD28 transmembrane domain,which was coupled to a FcεRIγ signaling domain. The so constructedBCMA-CAR had a nucleic acid sequence of SEQ ID NO:46.

BCMA expression was confirmed as is shown in the exemplary results ofFIG. 50 , and CAR mediated cytotoxicity was demonstrated against targetcells as is shown in FIG. 51 . Similarly, as can be seen from theresults in FIG. 52 , recombinant cells had significant ADCC usingrituximab as antibody against the target cells.

Example 17: GD2-CAR with FcεRIγ Signaling Domain

In this example, the inventors constructed a 1^(st) generation CARs witha FcεRIγ signaling domain that included an anti-GD2 scFv coupled to aCD8 hinge, that in turn was coupled to a CD28 transmembrane domain,which was coupled to a FcεRIγ signaling domain. The so constructedGD2-CAR had a nucleic acid sequence of SEQ ID NO:47.

Example 18: FAP-CAR with FcεRIγ Signaling Domain

In this example, the inventors constructed a 1^(st) generation CARs witha FcεRIγ signaling domain that included an anti-FAP scFv coupled to aCD8 hinge, that in turn was coupled to a CD28 transmembrane domain,which was coupled to a FcεRIγ signaling domain. The so constructedFAP-CAR had a nucleic acid sequence of SEQ ID NO:48. Expression of theFAP-CAR is shown in the data of FIG. 57 , and FAP.CAR cytotoxicity isdemonstrated on target cells in the results of FIG. 58 .

Example 19: CSPG-4-CAR with FcεRIγ Signaling Domain

In this example, the inventors constructed a 1^(st) generation CARs witha FcεRIγ signaling domain that included an anti-CSPG-4 scFv coupled to aCD8 hinge, that in turn was coupled to a CD28 transmembrane domain,which was coupled to a FcεRIγ signaling domain. The so constructedCSPG-4-CAR had a nucleic acid sequence of SEQ ID NO:52. Expression ofthe CSPG-4-CAR was confirmed with FACS analysis and exemplary resultsare shown in FIG. 59 . Thusly constructed cells also exhibitedsignificant cytotoxicity as is shown in the exemplary data of FIG. 60 .

Example 20: CD20-CAR with FcεRIγ Signaling Domain

In this example, the inventors constructed a 1^(st) generation CARs witha FcεRIγ signaling domain that included an anti-CD20 scFv coupled to aCD8 hinge, that in turn was coupled to a CD28 transmembrane domain,which was coupled to a FcεRIγ signaling domain. The so constructedCD20-CAR had a nucleic acid sequence of SEQ ID NO:51.

Expression of the CD20 CAR in NK-92 cells is shown in the results ofFIG. 25 . As can be readily seen, CD20.CAR is expressed strongly in thevast majority of recombinant cells (along with CD16 from the linearizedDNA as noted above). FIG. 26 depicts exemplary results for cytotoxicityof the CD20.CAR NK cells against CD20⁺ target cells.

Example 21: CD19-CAR with FcεRIγ Signaling Domain

In this example, the inventors used the 1^(st) generation CARs asdescribed above having a FcεRIγ signaling domain that included ananti-CD19 scFv coupled to a CD8 hinge, that in turn was coupled to aCD28 transmembrane domain, which was coupled to a FcεRIγ signalingdomain and transfected NK-92cells with linearized DNA for functionaltesting.

Functionality of the so constructed CD19.CAR-t-haNK cells was testedagainst K562 cells for determination of general cytotoxicity using astandard cytotoxicity assay and exemplary results are shown in FIG. 15 .As can be readily seen, the CD19.CAR-t-haNK cells expressing the CARwith the FcεRIγ signaling domain exhibited significant cytotoxicityagainst the K562 target cells. In a further set of experiments, targetspecific cytotoxicity was determined using SUP-B15 cells in comparisonwith aNK cells as control, and exemplary results are shown in FIG. 16 .Once more, CD19.CAR-t-haNK cells expressing the CAR with the FcεRIγsignaling domain exhibited significant and target specific cytotoxicity.In yet another set of experiments, target specific ADCC was determinedusing SKBr3 cells using Herceptin and Rituxan as antibodies, andexemplary results are shown in FIG. 17 . Again, CD19.CAR-t-haNK cellsexpressing the CAR with the FcεRIγ signaling domain exhibitedsignificant antibody and target specific ADCC.

FIG. 21 exemplarily illustrates CD19.CAR expression from linearized DNAthat included a segment encodingCD16 and IL-2^(ER) in NK-92 cells versuscontrol. As can be seen form FIG. 25 , the expression was very strongacross the vast majority of cells. Additional results for naturalcytotoxicity of CD19.CAR t-haNK cells against K562 cells and targetedcytotoxicity against SUP-B15 cells are depicted in FIG. 22 and FIG. 23 .Exemplary further results for ADCC of CD19.CAR t-haNK cells againstSUP-B15CD19^(KO)/CD20⁺ cells are shown in FIG. 24 .

Example 22: Anti-Tumor Activity of PD-L1-Targeting t-haNK Cells in HumanXenograft Models in NSG Mice

MDA-MB-231 and HCC827 were used as validated xenograft models that arePDL1 positive, and efficacy of PDL1 t-haNK cells in varied formulations,dosing levels, and dosing routes (IV and IT) was evaluated.

Animals: Animal type: NSG mice (JAX), females, 9-10 weeks old; Number ofanimals for MDA-MB-231 model: 24 (fresh cells), and for HCC827 model: 24(fresh cells)+6 (cryopreserved cells). Tumor model used the followingcell line: MDA-MB-231 (human breast adenocarcinoma) and HCC827 (humanlung adenocarcinoma), Route of inoculation was subcutaneous on bothflanks, and average tumor burden upon treatment initiation was forMDA-MB-231 about 100 mm3 and for HCC827 about 75-80 mm3.

Treatment articles: Anti-PD-L1 t-haNK, freshly prepared, irradiated, ata concentration: 5E7 cells/mL or 2E7 cells/mL; Vehicle control wasX-VIVO™ 10 medium; Method of administration was IV and IT as noted.Dosage for IV NK dosing was 1E7 cells/dose in 200 μL (Freshly preparedcells), 4E6 cells/dose in 200 μL (Cryopreserved cells); for IT NK dosing(fresh cells only) dose was 2.5E6 cells/tumor/dose in 50 μL. Dosingfrequency was Twice a week (M/Th or T/F) for 4 consecutive weeks, andfirst day of dosing was defined as Day 1.

Study design for MDA-MB-231 is in Table 3 below (This study was ended onDay 27, when some animals in Groups A, C and D had reached combinedtumor volume of >2000 mm3)

TABLE 2 NK NK Tumor Fresh or Cell dosing Treatment Dosing Group N modelTreatment Frozen Dose route Regimen Volume A 6 MDA-MB-231 Vehicle / / IVBIW × 4 200 μL SC, weeks B 6 bilateral PD-L1 Fresh   1E7 IV BIW × 4 200μL 1 × 10⁶ t-haNK weeks C 6 Vehicle / / IT BIW × 4  50 μL weeks D 6PD-L1 Fresh 2.5E6 IT BIW × 4  50 μL t-haNK weeks

Study design for HCC827 is in Table 4 below (This study was ended on Day29, when surviving animals were re-purposed and transferred to anotherstudy).

TABLE 3 NK NK Tumor Fresh or Cell dosing Treatment Dosing Group N modelTreatment Frozen Dose route Regimen Volume A 6 HCC827 Vehicle / / IV BIW× 4 200 μL SC, weeks B 6 bilateral PD-L1 Fresh   1E7 IV BIW × 4 200 μL 1× 10⁶ t-haNK weeks C 6 Vehicle / / IT BIW × 4  50 μL weeks D 6 PD-L1Fresh 2.5E6 IT BIW × 4  50 μL t-haNK weeks Pilot 6 PD-L1 Frozen   4E6 IVBIW × 4 200 μL t-haNK weeks

Results: Freshly prepared PD-L1 t-haNK cells (IE7 cells/dose) led tomarked and long-lasting tumor growth inhibition in both MDA-MB-231 andHCC827 models

MDA-MB-231: tumor stasis: TGI on Day 16: 84% (peak); TGI on Day 26: 79%(last measurement).

HCC827: tumor regression: TGI on Day 16: 120% (peak); TGI on Day 29: 84%(study end).

Cryopreserved PDL1 t-haNK cells (4E6 cells/dose) also showedstatistically significant efficacy in suppressing tumor growth comparedto X-VIVO™ 10 media: TGI on Day 26: 60% (peak), and TGI on Day 29: 40%(study end).

Freshly prepared PDL1 t-haNK cells (IE7 cells/dose) also led tosignificant reduction of metastatic disease burden in the MDA-MB-231model as shown in Table 5 below.

TABLE 4 Macroscopic lesions Group Mouse found in: Overall Summary A 1Liver, lungs 100% animals (vehicle) 2 Ax LNs, liver, lungs developedmetastases 3 Ax LN (left), liver, lungs in multiple organs 4 Liver,lungs 5 Ax LNs, spleen, liver, lungs 6 Ax LNs, liver, lungs B (PD-L1 1None 50% developed t-haNK) 2 Lungs metastasis; all 3 Ax LNs single-organfindings 4 None 5 Ax LN (left) 6 None

The number of visible nodules in liver was in vehicle: 29±9, in thePD-L1 t-haNK group: 0 (P=0.0116 by unpaired 2-tailed t test).

Based on the experiments performed, IV dosing of freshly prepared PD-L1t-haNK cells at the dosing level of 1E7 cells/dose, twice a week for 4weeks, showed marked anti-tumor efficacy in both of the subcutaneousxenograft models tested: The treatment resulted in tumor stasis inMDA-MB-231 tumor-bearing mice, with a peak TGI of 84% on Day 16 and anend-of-study TGI of 79% (P<0.0001 for both time points by 2-way ANOVAfollowed by multiple comparison by Tukey test), and tumor regression inthe HCC827 model, with a peak TGI of 120% on Day 16 and an end-of-studyTGI of 84% (P<0.0001). IV dosing of cryopreserved PD-L1 t-haNK cells atthe dosing level of 4E6 cells/dose, twice a week for 4 weeks, alsoshowed significant therapeutic efficacy in the HCC827 tumor model,reaching a peak TGI of 60% (P<0.0001), and an end-of-study TGI of 40%(P<0.01). IT dosing of freshly prepared PD-L1 t-haNK cells at the dosinglevel of 2.5E6 cells/dose/tumor, twice a week for 4 weeks, effectivelysuppressed the growth of HCC827 tumors, resulting in a peak TGI of 70%on Day 20 and an end-of-study TGI of 49% (P<0.001).

Significant adverse reactions were observed for animals that received IVadministrations of freshly prepared PD-L1 t-haNK cells (1E7 cells/dose).In contrast to freshly prepared PD-L1 t-haNK cells, cryopreserved cells(dosed at a lower level of 4E6 cells/dose) proved to be safe to theanimals after IV administrations. PD-L1 t-haNK cells demonstratedremarkable efficacy in the two subcutaneous tumor models. Cryopreservedcells dosed at the lower 4E6 cells/dose level, also showed significantefficacy in suppressing tumor growth, and proved to be safe for theanimals.

Of course, it should be recognized that for all nucleic acid sequencesprovided herein the corresponding encoded proteins are also expresslycontemplated herein. Likewise, for all amino acid sequences,corresponding nucleic acids sequences are also contemplated herein (withany codon usage).

All patent applications, publications, references, and sequenceaccession numbers cited in the present specification are herebyincorporated by reference in their entirety.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

It is understood that all numerical values described herein (e.g., pH,temperature, time, concentration, amounts, and molecular weight,including ranges) include normal variation in measurements encounteredby one of ordinary skill in the art. Thus, numerical values describedherein include variation of +/−0.1 to 10%, for example, +/−0.1%, 0.5%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. It is to be understood,although not always explicitly stated, that all numerical designationsmay be preceded by the term “about.” Thus, the term about includesvariation of +/−0.1 to 10%, for example, +/−0.1%, 0.5%, 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, or 10% of the numerical value. It is also to beunderstood, although not always explicitly stated, that the reagentsdescribed herein are merely exemplary and that equivalents of such areknown in the art.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein include the end points of the range, and includeall values between the end points of the range. All ranges disclosedherein also encompass any and all possible subranges and combinations ofsubranges thereof. Any listed range can be easily recognized assufficiently describing and enabling the same range being broken downinto at least equal halves, thirds, quarters, fifths, tenths, etc. As anon-limiting example, each range discussed herein can be readily brokendown into a lower third, middle third and upper third, etc. As will alsobe understood by one skilled in the art all language such as “up to,”“at least,” and the like, include the number recited and refer to rangeswhich can be subsequently broken down into subranges as discussed above.Finally, as will be understood by one skilled in the art, a rangeincludes each individual member. Thus, for example, a group having 1-3cells refers to groups having 1, 2, or 3 cells. Similarly, a grouphaving 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and soforth.

It is also to be understood, although not always explicitly stated, thatthe reagents described herein are merely exemplary and that equivalentsof such are known in the art.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The term “comprising” is intended to mean that the compositions andmethods include the recited elements, but not excluding others.“Consisting essentially of,” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. For example, a composition consistingessentially of the elements as defined herein would not exclude otherelements that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. “Consisting of” shall meanexcluding more than trace amount of other ingredients and substantialmethod steps recited. Embodiments defined by each of these transitionterms are within the scope of this disclosure.

As used herein, “immunotherapy” refers to the use of NK-92 cells,modified or unmodified, naturally occurring or modified NK cell orT-cell, whether alone or in combination, and which are capable ofinducing cytotoxicity when contacting a target cell.

As used herein, “natural killer (NK) cells” are cells of the immunesystem that kill target cells in the absence of a specific antigenicstimulus, and without restriction according to major histocompatibilitycomplex (MHC) class. Target cells may be tumor cells or cells harboringa virus. NK cells are characterized by the presence of CD56 and theabsence of CD3 surface markers.

The term “endogenous NK cells” is used to refer to NK cells derived froma donor (or the patient), as distinguished from the NK-92 cell line.Endogenous NK cells are generally heterogeneous populations of cellswithin which NK cells have been enriched. Endogenous NK cells may beintended for autologous or allogeneic treatment of a patient.

The term “NK-92” refers to natural killer cells derived from the highlypotent unique cell line described in Gong et al. (1994), rights to whichare owned by NantKwest (hereafter, “NK-92™ cells”). The immortal NK cellline was originally obtained from a patient having non-Hodgkin'slymphoma. Unless indicated otherwise, the term “NK-92™” is intended torefer to the original NK-92 cell lines as well as NK-92 cell lines thathave been modified (e.g., by introduction of exogenous genes). NK-92™cells and exemplary and non-limiting modifications thereof are describedin U.S. Pat. Nos. 7,618,817; 8,034,332; 8,313,943; 9,181,322; 9,150,636;and published U.S. application Ser. No. 10/008,955, all of which areincorporated herein by reference in their entireties, and include wildtype NK-92™ NK-92™-CD16, NK-92™-CD16-γ, NK-92™-CD16-ζ,NK-92™-CD16(F176V), NK-92™MI, and NK-92™CI. NK-92 cells are known topersons of ordinary skill in the art, to whom such cells are readilyavailable from NantKwest, Inc.

The term “aNK” refers to an unmodified natural killer cells derived fromthe highly potent unique cell line described in Gong et al. (1994),rights to which are owned by NantKwest (hereafter, “aNK™ cells”). Theterm “haNK” refers to natural killer cells derived from the highlypotent unique cell line described in Gong et al. (1994), rights to whichare owned by NantKwest, modified to express CD16 on the cell surface(hereafter, “CD16+ NK-92™ cells” or “haNK® cells”). In some embodiments,the CD16+NK-92™ cells comprise a high affinity CD16 receptor on the cellsurface. The term “taNK” refers to natural killer cells derived from thehighly potent unique cell line described in Gong et al. (1994), rightsto which are owned by NantKwest, modified to express a chimeric antigenreceptor (hereafter, “CAR-modified NK-92™ cells” or “taNK® cells”). Theterm “t-haNK” refers to natural killer cells derived from the highlypotent unique cell line described in Gong et al. (1994), rights to whichare owned by NantkWest, modified to express CD 16 on the cell surfaceand to express a chimeric antigen receptor (hereafter, “CAR-modifiedCD16+NK-92™ cells” or “t-haNK™ cells”). In some embodiments, the t-haNK™cells express a high affinity CD16 receptor on the cell surface.

A “modified NK-92 cell” refers to an NK-92 cell that expresses anexogenous gene or protein, such as an Fc receptor, a CAR, a cytokine(such as IL-2 or IL-15), and/or a suicide gene. In some embodiments, themodified NK-92 cell comprises a vector that encodes for a transgene,such as an Fc receptor, a CAR, a cytokine (such as IL-2 or IL-15),and/or a suicide gene. In one embodiment, the modified NK-92 cellexpresses at least one transgenic protein.

As used herein, “non-irradiated NK-92 cells” are NK-92 cells that havenot been irradiated. Irradiation renders the cells incapable of growthand proliferation. It is envisioned that the NK-92 cells will beirradiated at the treatment facility or some other point prior totreatment of a patient, since the time between irradiation and infusionshould be no longer than four hours in order to preserve optimalactivity. Alternatively, NK-92 cells may be prevented from proliferatingby another mechanism.

As used herein, “inactivation” of the NK-92 cells renders them incapableof growth. Inactivation may also relate to the death of the NK-92 cells.It is envisioned that the NK-92 cells may be inactivated after they haveeffectively purged an ex vivo sample of cells related to a pathology ina therapeutic application, or after they have resided within the body ofa mammal a sufficient period of time to effectively kill many or alltarget cells residing within the body. Inactivation may be induced, byway of non-limiting example, by administering an inactivating agent towhich the NK-92 cells are sensitive.

As used herein, the terms “cytotoxic” and “cytolytic,” when used todescribe the activity of effector cells such as NK-92 cells, areintended to be synonymous. In general, cytotoxic activity relates tokilling of target cells by any of a variety of biological, biochemical,or biophysical mechanisms. Cytolysis refers more specifically toactivity in which the effector lyses the plasma membrane of the targetcell, thereby destroying its physical integrity. This results in thekilling of the target cell. Without wishing to be bound by theory, it isbelieved that the cytotoxic effect of NK-92 cells is due to cytolysis.

The term “kill” with respect to a cell/cell population is directed toinclude any type of manipulation that will lead to the death of thatcell/cell population.

The term “Fc receptor” refers to a protein found on the surface ofcertain cells (e.g., natural killer cells) that contribute to theprotective functions of the immune cells by binding to part of anantibody known as the Fc region. Binding of the Fc region of an antibodyto the Fc receptor (FcR) of a cell stimulates phagocytic or cytotoxicactivity of a cell via antibody-mediated phagocytosis orantibody-dependent cell-mediated cytotoxicity (ADCC). FcRs areclassified based on the type of antibody they recognize. For example,Fc-gamma receptors (FCγR) bind to the IgG class of antibodies. FCγRIII-A(also called CD16; SEQ ID NO:34) is a low affinity Fc receptor bind toIgG antibodies and activate ADCC. FCγRIII-A are typically found on NKcells. NK-92 cells do not express FCγRIII-A. Fc-epsilon receptors (FcR)bind to the Fc region of IgE antibodies.

The term “chimeric antigen receptor” (CAR), as used herein, refers to anextracellular antigen-binding domain that is fused to an intracellularsignaling domain. CARs can be expressed in T cells or NK cells toincrease cytotoxicity. In general, the extracellular antigen-bindingdomain is a scFv that is specific for an antigen found on a cell ofinterest. A CAR-expressing NK-92 cell is targeted to cells expressingcertain antigens on the cell surface, based on the specificity of thescFv domain. The scFv domain can be engineered to recognize any antigen,including tumor-specific antigens and virus-specific antigens. Forexample, CD19CAR recognizes CD19, a cell surface marker expressed bysome cancers.

The term “tumor-specific antigen” as used herein refers to antigens thatare present on a cancer or neoplastic cell but not detectable on anormal cell derived from the same tissue or lineage as the cancer cell.Tumor-specific antigens, as used herein, also refers to tumor-associatedantigens, that is, antigens that are expressed at a higher level on acancer cell as compared to a normal cell derived from the same tissue orlineage as the cancer cell.

The term “virus-specific antigen” as used herein refers to antigens thatare present on a virus-infected cell but not detectable on a normal cellderived from the same tissue or lineage as the virus-infected cell. Inone embodiment, a virus-specific antigen is a viral protein expressed onthe surface of an infected cell.

The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” areused interchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probesand primers. A polynucleotide can comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure can be imparted before or after assembly ofthe polynucleotide. The sequence of nucleotides can be interrupted bynon-nucleotide components. A polynucleotide can be further modifiedafter polymerization, such as by conjugation with a labeling component.The term also refers to both double- and single-stranded molecules.Unless otherwise specified or required, any embodiment of this inventionthat is a polynucleotide encompasses both the double-stranded form andeach of two complementary single-stranded forms known or predicted tomake up the double-stranded form.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences.

As used herein, “percent identity” refers to sequence identity betweentwo peptides or between two nucleic acid molecules. Percent identity canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare identical at that position. Homologous nucleotide sequences includethose sequences coding for naturally occurring allelic variants andmutations of the nucleotide sequences set forth herein. Homologousnucleotide sequences include nucleotide sequences encoding for a proteinof a mammalian species other than humans. Homologous amino acidsequences include those amino acid sequences which contain conservativeamino acid substitutions and which polypeptides have the same bindingand/or activity. In some embodiments, a homologous amino acid sequencehas no more than 15, nor more than 10, nor more than 5 or no more than 3conservative amino acid substitutions. In some embodiments, a nucleotideor amino acid sequence has at least 60%, at least 65%, at least 70%, atleast 80%, or at least 85% or greater percent identity to a sequencedescribed herein. In some embodiments, a nucleotide or amino acidsequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%identity to a sequence described herein. Percent identity can bedetermined by, for example, the Gap program (Wisconsin Sequence AnalysisPackage, Version 8 for UNIX, Genetics Computer Group, UniversityResearch Park, Madison Wis.), using default settings, which uses thealgorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).Algorithms suitable for determining percent sequence identity includethe BLAST and BLAST 2.0 algorithms, which are described in Altschul etal. (Nuc. Acids Res. 25:3389-402, 1977), and Altschul et al. (J. Mol.Biol. 215:403-10, 1990), respectively. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (see the internet at ncbi.nlm.nih.gov). TheBLAST algorithm parameters W, T, and X determine the sensitivity andspeed of the alignment. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc.Natl. Acad. Sci. USA 89:10915, 1989) alignments (B) of 50, expectation(E) of 10, M=5, N=−4.

In some embodiments, a nucleic acid sequence is codon optimized forexpression in a particular species, for example, a mouse sequence can becodon optimized for expression in humans (expression of the proteinencoded by the codon-optimized nucleic acid sequence). Thus, in someembodiments, a codon-optimized nucleic acid sequence has at least 60%,at least 65%, at least 70%, at least 80%, or at least 85% or greaterpercent identity to a nucleic acid sequence described herein. In someembodiments, a codon-optimized nucleic acid sequence acid sequence hasat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity toa sequence described herein.

The term “express” refers to the production of a gene product (e.g., aprotein). The term “transient” when referring to expression means apolynucleotide is not incorporated into the genome of the cell. The term“stable” when referring to expression means a polynucleotide isincorporated into the genome of the cell, or a positive selection marker(i.e., an exogenous gene expressed by the cell that confers a benefitunder certain growth conditions) is utilized to maintain expression ofthe transgene.

The term “cytokine” or “cytokines” refers to the general class ofbiological molecules which affect cells of the immune system. Exemplarycytokines include but are not limited to interferons and interleukins(IL)—in particular IL-2, IL-12, IL-15, IL-18 and IL-21. In preferredembodiments, the cytokine is IL-2.

As used herein, the term “vector” refers to a non-chromosomal nucleicacid comprising an intact replicon such that the vector may bereplicated when placed within a permissive cell, for example by aprocess of transformation. A vector may replicate in one cell type, suchas bacteria, but have limited or no ability to replicate in anothercell, such as mammalian cells. Vectors may be viral or non-viral.Exemplary non-viral vectors for delivering nucleic acid include nakedDNA; DNA complexed with cationic lipids, alone or in combination withcationic polymers; anionic and cationic liposomes; DNA-protein complexesand particles comprising DNA condensed with cationic polymers such asheterogeneous polylysine, defined-length oligopeptides, and polyethyleneimine, in some cases contained in liposomes; and the use of ternarycomplexes comprising a virus and polylysine-DNA. In one embodiment, thevector is a viral vector, e.g. adenovirus. Viral vectors are well knownin the art.

As used herein, the term “targeted,” when referring to proteinexpression, is intended to include, but is not limited to, directingproteins or polypeptides to appropriate destinations in the cell oroutside of it. The targeting is typically achieved through signalpeptides or targeting peptides, which are a stretch of amino acidresidues in a polypeptide chain. These signal peptides can be locatedanywhere within a polypeptide sequence, but are often located on theN-terminus. Polypeptides can also be engineered to have a signal peptideon the C-terminus. Signal peptides can direct a polypeptide forextracellular section, location to plasma membrane, golgi, endosomes,endoplasmic reticulum, and other cellular compartments. For example,polypeptides with a particular amino acid sequence on their C-terminus(e.g., KDEL) are retained in the ER lumen or transported back the ERlumen.

As used herein, the term “target,” when referring to targeting of atumor, refers to the ability of NK-92 cells to recognize and kill atumor cell (i.e., target cell). The term “targeted” in this contextrefers, for example, to the ability of a CAR expressed by the NK-92 cellto recognize and bind to a cell surface antigen expressed by the tumor.

As used herein, the term “transfect” refers to the insertion of nucleicacid into a cell. Transfection may be performed using any means thatallows the nucleic acid to enter the cell. DNA and/or mRNA may betransfected into a cell. Preferably, a transfected cell expresses thegene product (i.e., protein) encoded by the nucleic acid.

The term “suicide gene” refers to a transgene that allows for thenegative selection of cells expressing that transgene. A suicide gene isused as a safety system, allowing the cells expressing the gene to bekilled by introduction of a selective agent. A number of suicide genesystems have been identified, including the herpes simplex virusthymidine kinase (TK) gene, the cytosine deaminase gene, thevaricella-zoster virus thymidine kinase gene, the nitroreductase gene,the Escherichia coli gpt gene, and the E. coli Deo gene (see also, forexample, Yazawa K, Fisher W E, Brunicardi F C: Current progress insuicide gene therapy for cancer. World J. Surg. 2002 July; 26(7):783-9).In one embodiment, the suicide gene is the thymidine kinase (TK) gene.The TK gene may be a wild-type or mutant TK gene (e.g., tk30, tk75,sr39tk). Cells expressing the TK protein can be killed usingganciclovir.

What is claimed is:
 1. A genetically modified NK-92 cell, comprising: arecombinantly expressed cytokine; a recombinantly expressed CD16; amembrane bound chimeric antigen receptor (CAR) that comprises a FcεRIγsignaling domain, wherein the CAR has at least 90% sequence identity toa polypeptide encoded by a nucleic acid having sequence SEQ ID NO:41,including 100% identity in the binding domain comprised therein, whereinthe binding domain comprises a scFv portion that binds to CD123.
 2. Thegenetically modified NK-92 cell of claim 1, wherein the recombinantlyexpressed cytokine is IL-2, optionally comprising an endoplasmicretention sequence.
 3. The genetically modified NK-92 cell of claim 1,wherein the recombinantly expressed cytokine is IL-15, optionallycomprising an endoplasmic retention sequence.
 4. The geneticallymodified NK-92 cell of claim 1, wherein the recombinantly expressed CD16has at least 80% sequence identity to the amino acid sequence of SEQ IDNO:
 35. 5. The genetically modified NK-92 cell of claim 1, wherein theFcεRIγ signaling domain has at least 90% sequence identity to the aminoacid sequence of SEQ ID NO:1.
 6. The genetically modified NK-92 cell ofclaim 1, wherein the FcεRIγ signaling domain has an amino acid sequenceof SEQ ID NO:1.
 7. The genetically modified NK-92 cell of claim 1,wherein the genetically modified NK cell comprises a tricistronicnucleic acid sequence comprising a sequence encoding the recombinantlyexpressed cytokine, a sequence encoding the recombinantly expressedCD16, and a sequence encoding the recombinantly expressed CAR.
 8. Thegenetically modified NK-92 cell of claim 7, wherein the tricistronicnucleic acid sequence is integrated into the genome of the NK-92 cell.9. A recombinant nucleic acid encoding a CAR that has at least 90%sequence identity to a polypeptide encoded by a nucleic acid havingsequence SEQ ID NO:41, including 100% identity in a binding domaincomprised therein, wherein the binding domain comprises a scFv portionthat binds to CD123.
 10. The recombinant nucleic acid of claim 9,wherein the recombinant nucleic acid encodes a CAR that has at least 95%sequence identity to the polypeptide encoded by a nucleic acid havingsequence SEQ ID NO:41.
 11. The recombinant nucleic acid of claim 9,wherein the recombinant nucleic acid has a nucleic acid having sequenceSEQ ID NO:41.
 12. A method of treating cancer in a patient in needthereof, comprising administering to the patient a therapeuticallyeffective amount of the genetically modified NK cells of claim 1,thereby treating the cancer.
 13. The method of claim 12 furthercomprising a step of administering at least one additional therapeuticentity selected from the group consisting of a viral cancer vaccine, abacterial cancer vaccine, a yeast cancer vaccine, N-803, an antibody, astem cell transplant, and a tumor targeted cytokine.
 14. The method ofclaim 12, wherein the cancer is selected from leukemia, acutelymphocytic leukemia, acute myelocytic leukemia, chronic leukemias,chronic myelocytic (granulocytic) leukemia, chronic lymphocyticleukemia, polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin'sdisease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chaindisease, solid tumors including, but not limited to, sarcomas andcarcinomas such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovariancancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilm's tumor, cervical cancer, testicular tumor, lungcarcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma.